Anti cd37 antibodies

ABSTRACT

Chimeric and humanized anti-CD37 antibodies and pharmaceutical compositions containing them are useful for the treatment of B cell malignancies and autoimmune and inflammatory diseases that involve B cells in their pathology.

The present invention relates to immunotherapies that are based on Bcell depletion. In particular, the present invention relates toanti-CD37 antibody molecules for use in such therapies, e.g. in thetreatment of B cell malignancies and autoimmune conditions.

Immunotherapy using monoclonal antibodies (mAbs) has been emerging as asafe and selective method for the treatment of cancer and otherdiseases. In particular, the role of monoclonal antibodies in therapiesthat are based on B cell depletion, e.g. in the treatment of B cellmalignancies, has expanded since the introduction of rituximab(Rituxan®), an antibody that is directed against the CD20 antigen on theB cell surface. Numerous studies have confirmed the efficacy ofrituximab as a single agent and in combination therapy in low-grade NHL(Hiddemann et al., 2005a; Hiddemann et al., 2005b; Hainsworth 2004;McLaughlin et al., 1998), mantle cell lymphoma (Forstpointner et al.,2004; Kahl et al., 2006; Foran et al., 2000; Howard et al., 2002;Romaguera et al., 2005), diffuse large cell lymphoma (DLCL) (Coiffier etal., 1998; Feugier et al., 2005), and Burkitt leukemia/lymphoma (Thomaset al., 2006). However, only a subset of patients respond to therapy andthe majority of those eventually relapse following rituximab treatment.Therefore, new therapeutic targets on B cells have been sought that arepotentially more effective than CD20 for therapy of B cell malignancies(Zhao et al., 2007). The CD37 antigen is a cell surface antigen thathas, to date, not been considered as a target for B cell malignancies tothe same extent as the B cell antigen CD20.

CD37, a member of the tetraspanin superfamily, is a heavily glycosylatedcell surface molecule with four transmembrane domains and twoextracellular loops. CD37 is almost exclusively expressed on mature Bcells, with highest expression levels on peripheral blood B cells,reduced levels on plasma cells and non-detectable levels on CD10+precursor B cells in the bone marrow. Low level expression of CD37 hasalso been reported on resting and activated T cells, granulocytes, andmonocytes. In B cell neoplasm, CD37 expression is mainly observed inaggressive non-Hodgkin's lymphoma (NHL) and chronic lymphoid leukemia(CLL). High level of CD37 expression is also found on mantle celllymphoma (MCL). This expression pattern makes CD37 an attractive targetfor antibody-mediated cancer therapy.

CD37 was first described in 1986 and characterized by the murinemonoclonal antibody MB-1 (Link et al., 1986).

The physiological role of CD37 is unknown. Mice deficient for CD37display no changes in development and cellular composition of lymphoidorgans, but have reduced levels of IgG1 and attenuated T cell mediatedimmune reactions (Knobeloch et al., 2000). Studies with CD37^(−/−) Tcells suggest a role for CD37 in T cell proliferation (van Spriel etal., 2004).

CD37 expression on malignant B cells of various diseases has beenreported. CD37 is expressed in the majority of mature B cellmalignancies like Burkitt lymphoma, follicular lymphoma and lymphocyticlymphoma (Link et al., 1986). High levels of CD37 expression have beenobserved in hairy cell leukemia and in samples of patients with chroniclymphocytic leukemia (CLL) and different subtypes of non-Hodgkin'slymphoma (NHL) including mantle cell lymphoma (MCL) (Schwartz-Albiez etal., 1988; Barrena et al., 2005). One report utilizing antibody microarray for immunophenotyping claims CD37 to be a good discriminatorbetween malignant CLL cells (high CD37 expression) versus normalperipheral blood (PB) lymphocytes (low CD37 expression) (Belov et al.,2001).

Binding of a CD37-specific mAb to cancer cells may trigger variousmechanisms of action: First, after the antibody binds to theextracellular domain of the CD37 antigen, it may activate the complementcascade and lyse the targeted cell. Second, an anti-CD37 antibody maymediate antibody-dependent cell-mediated cytotoxicity (ADCC) to thetarget cell, which occurs after the Fc portion of the bound antibody isrecognized by appropriate receptors on cytotoxic cells of the immunesystem.

Third, the antibody may alter the ability of B cells to respond toantigen or other stimuli. Finally, anti-CD37 antibody may initiateprogrammed cell death (apoptosis).

Anti-CD37 mAb MB-1 was evaluated in two radio-immunotherapy trials inB-NHL patients (B-cell non-Hodgkin's lymphoma; Press et al., 1989;Kaminski et al., 1992). Therapeutic doses of ¹³¹I-MB-1 were administeredto 6 relapsed NHL patients in one trial and all 6 patients achieved aclinical complete remission (CR) with a median duration of 7 months. Ofnote, two of the six patients showed clinical regressions already afteradministration of only the tracer dose of MB-1 suggesting a directanti-tumor effect of the antibody itself. In the second trialradiolabeled MB-1 was applied for the treatment of refractory NHLpatients and resulted in 3 out of 9 evaluable patients with objectiveresponses of limited duration (Kaminski et al., 1992). In both trials arapid and transient depletion of peripheral B cells after injection ofthe trace labeled MB-1 antibody dose was reported. These observationssupport the conclusion that MB-1 exerts a cytotoxic activity on its own.In summary, these clinical trials underscore the feasibility ofCD37-targeting for B-cell malignancies and point to a potential clinicalrelevance of anti-CD37 therapy.

There is experimental evidence with a CD37 specific antibody-like singlechain molecule (“Small Modular ImmunoPharmaceutical”, SMIP) thattreatment with that molecule induces apoptosis in vitro and delaysBurkitt lymphoma growth in a xenograft model in vivo. Anti-apoptoticactivity of the recombinant anti-CD37 SMIP Tru16.4 from Trubion wasdescribed recently (Zhao et al., 2004). Tru 16.4 inducedcaspase-independent apoptosis on primary CLL cells from tumor patients.Induction of apoptosis on these cells was greater than that of Rituximaband comparable to that of Alemtuzumab, a CD52 antagonist. The degree ofapoptosis induction was directly proportionate to CD37 cell surfaceexpression and could be further enhanced by cross-linking with ananti-human IgG antibody. A correlation of CD37 expression and ADCC wasdemonstrated on cell lines in vitro. In a Burkitt lymphoma mouse model(Raji) treatment with anti-CD37 scFv revealed therapeutic efficacy (Zhaoet al., 2007). These data provide first evidence that CD37-targeting isa promising approach for targeted anti-tumor therapy by induction ofapoptosis and ADCC.

In conclusion, it has been shown that the CD37 antigen is frequentlyexpressed on tumor cells in several human B cell malignancies and onmature normal B lymphocytes and that anti-CD37-based therapy may be apromising approach for treating B cell malignancies. The depletion ofCD37-positive normal B cells is not considered critical since clinicaldata from numerous patients show that even prolonged depletion of Bcells up to 6 months with an anti-CD20 mAb does not significantly reduceIgG serum levels or increases the risk of infections (Van der Kolk etal., 2002).

Although the anti-CD37 antibodies or antibody-like molecules describedabove (MB-1 and SMIP Tru16.4) have shown anti-tumor efficacy in B-cellmalignancies and the potential to target CD37, there is a need foralternate anti-CD37 inhibitors to improve therapies based on B-celldepletion.

SUMMARY OF INVENTION

It was an object of the invention to provide novel CD37 antagonists forthe treatment of B cell malignancies and other disorders which respondto the depletion of CD37 positive B cells.

Furthermore, it was an object of the invention to provide anti-CD37antibodies with improved effector functions. In particular, theinventors sought to provide anti-CD37 mABs with antibody-dependentcell-mediated cytotoxicity (ADCC).

To solve the problem underlying the invention, a murine monoclonalanti-CD37 antibody was used as a starting antibody for generatingchimeric and humanized anti-CD37 antibodies that are useful in humantherapy.

In a first aspect, the present invention provides an antibody moleculethat binds to human CD37 and that is derived from

-   -   a) a murine monoclonal antibody that is defined by        -   i. a variable heavy chain comprising the amino acid sequence            shown in SEQ ID NO: 2; and        -   ii. a variable light chain comprising the amino acid            sequence shown in SEQ ID NO:4, or from    -   b) a non-human antibody recognizing the same epitope of human        CD37 as the antibody defined in a) or recognizing an epitope        that is close to or overlaps with said epitope;        wherein said antibody molecule is a chimeric or a humanized        antibody.

As will be understood from the following, an antibody that is “derived”from another antibody, i.e. the starting antibody, means that saidantibody has been generated by modification of the starting antibody asdescribed below.

In a preferred embodiment, the antibody molecule is a chimeric orhumanized antibody molecule derived from the starting antibody definedin a). An antibody with a related sequence was designated G28.1 anddescribed in WO 2005/017148.

The starting antibody of category b) may, for example, be selected fromthe CD37-specific antibodies that characterized, like G28.1, the CD37antigen in the Third HLDA Workshop; these antibodies were designatedHD28, HH1, BI14, F97-3G6 (Ling and MacLennan, 1987). Other CD37-specificantibodies that have been described include RFB-7, Y29/55, MB-1, M-B371,M-B372 and IPO-24. According to Moldenhauer, 2000, and Schwartz-Albiezet al., 1988, all these antibodies (including G28.1) recognize the sameor an overlapping or close CD37 epitope. Schwartz-Albiez et al., 1988,indicates that the epitope is situated in the carbohydrate moiety ofCD37. A number of the above antibodies is commercially available, e.g.HH1 (SantaCruz), RFB-7 (Biodesign), Y29/55 (Biogenesis), M-B371 (BDBiosciences), M-B372 (SantaCruz) and IPO-24 (AbCam).

Other CD37-specific antibodies are S-B3 (Biosys), NMN46 (Chemicon), andICO-66 (Bioprobe). Whether an antibody recognizes the same epitope asG28.1 can be determined by competitive binding assays or by crossinhibition radioimmunoassays as described by Moldenhauer et al., 1987,and Moldenhauer, 2000.

By way of example, competitive binding may be determined in an ELISA,using plates coated with CD37 protein or CD37 peptides or with CD37positive cells (Cell ELISA) and measuring binding of biotinylatedantibody in the presence of a competitor candidate antibody. In thepresence of a competing antibody or antibody-derived fragment, thebinding of biotinylated G28.1 (or another antibody known to recognizethe same epitope) is reduced in the case that the antibodies recognize ashared epitope. To identify the G28.1 epitope peptide, fragments orshort polypeptides or recombinant proteins derived from the CD37sequence can be synthesized or produced and the binding of G28.1 to saidpeptides/polypeptides measured in an ELISA assay. Competitive bindingcan also be determined by FACS analysis, as described in the Examples.

An antibody defined in b) may be used in an analogous manner as G28.1 asa starting antibody for the generation of chimeric or humanized antibodymolecules.

A starting antibody of category b) may also be generated de novo byusing peptides or protein fragments containing the relevant epitope, orDNA molecules encoding such peptides/fragments, respectively forimmunization to obtain antibodies reactive with the same epitope asG28.1.

A starting antibody b) may also be obtained by immunization with wholecells carrying the relevant epitope; the thus obtained hybridoma cellsare then screened for competitive binding of the secreted antibodies.

The term “anti-CD37 antibody molecule” encompasses anti-CD37 antibodiesand anti-CD37 antibody fragments as well as conjugates with antibodymolecules. Antibodies include, in the meaning of the present invention,chimeric monoclonal and humanized monoclonal antibodies. The term“antibody”, which may interchangeably be used with “antibody molecule”,shall encompass complete immunoglobulins (as they are produced bylymphocytes and for example present in blood sera), monoclonalantibodies secreted by hybridoma cell lines, polypeptides produced byrecombinant expression in host cells, which have the binding specificityof immunoglobulins or monoclonal antibodies, and molecules which havebeen derived from such antibodies by modification or further processingwhile retaining their binding specificity.

In an embodiment of the invention, the anti-CD37 antibody molecule is achimeric antibody defined by

-   -   i) a variable heavy chain comprising the amino acid sequence        shown in SEQ ID NO: 2;    -   ii) a variable light chain comprising the amino acid sequence        shown in SEQ ID NO:4;    -   iii) constant heavy and light chains that are of human origin.

The construction and production of chimeric mouse/human antibodies iswell known in the art (Boulianne et al., 1984). The variable regions ofthe non-human antibody are typically linked to at least a portion(F_(C)) of the immunoglobulin constant region of a human immunoglobulin.Human constant region DNA sequences can be isolated in accordance withwell-known procedures from a variety of human cells, preferably fromimmortalized B cells (see Kabat et al., 1991; and WO 87/02671). Theantibody molecules may contain all or only a portion of the constantregion as long as they exhibit specific binding to the CD37 antigen andthe Fc receptors. The choice of the type and length of the constantregion depends on whether effector functions like complement fixation orantibody-dependent cell-mediated toxicity are desired, and on thedesired pharmacological properties of the antibody molecule.

In certain embodiments, the antibody molecule of the invention is achimeric CD37-specific antibody that has the heavy chain variable regionof a non-human antibody defined in a) or b) fused to the human heavychain constant region IgG1 and the light chain variable region of anon-human antibody defined in a) or b) fused to the human light chainconstant region kappa.

In yet another embodiment, the antibody molecule is a chimericCD37-specific antibody that has the heavy chain variable region shown inSEQ ID NO:2 fused to the human heavy chain constant region IgG1 which isan IgG1 molecule with the sequence shown in SEQ ID NO:24 (coding DNAsequence: SEQ ID NO: 23) or a mutated IgG1 molecule derived therefromand that has the light chain variable region shown in SEQ ID NO:4 fusedto the human light chain constant region kappa shown in SEQ ID NO:26(coding DNA sequence: SEQ ID NO: 25).

Other human constant regions for chimerizing a non-human startingantibody defined in a) or b) are available to the person skilled in theart, e.g. IgG2, IgG3, IgG4, IgA, IgE or IgM (instead of IgG1) or lambda(instead of kappa). The constant regions may also be chimeric, forexample, a heavy chain IgG1/IgG2 or IgG1/IgG3 chimera.

In certain embodiments of the invention, the anti-CD37 antibody moleculeis a humanized antibody that is defined by

-   -   i. CDRs contained within the variable heavy chain as shown in        SEQ ID NO:2 and by    -   ii. CDRs contained within the variable light chain as shown in        SEQ ID NO:4,    -   iii. frameworks supporting said CDRs that are derived from a        human antibody,    -   iv. constant heavy and light chains that are from a human        antibody.

Humanized forms of non-human (e.g. murine, rat or rabbit antibodies)antibodies are immunoglobulins, immunoglobulin chains or fragmentsthereof (such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-bindingmolecules with subsequences of antibodies) that contain minimalsequences derived from non-human immunoglobulin.

Humanized antibodies include human immunoglobulins (from the recipientantibody) in which residues from a complementarity determining region(CDR) of the recipient antibody are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues.

In the humanized antibodies of the invention, the sequences encodingCDRs of a non-human starting antibody defined in a) or b) have beengrafted into the respective genes of human immunoglobulin heavy andlight chains.

“Complementarity determining regions” (CDRs) of a monoclonal antibodyare understood to be those amino acid sequences involved in specificantigen binding according to Kabat et al., 1991, in connection withChothia and Lesk (1987). From the sequences of the variable regions asshown in SEQ ID NO:2 and SEQ ID NO:4, the CDR sequence can be routinelydetermined by searching the Kabat sequence database for sequencefeatures.

Techniques for obtaining humanized antibodies are routinely available tothe skilled person, they have been described, inter alia, in U.S. Pat.No. 5,225,539; U.S. Pat. No. 6,548,640; and U.S. Pat. No. 6,982,321.

Appropriate framework residues of the CDR-grafted antibody may bereverted to murine residues to improve binding affinity. As describedabove, from methods pertinent to the art, the expert knows how to obtainthe CDRs from a given non-human antibody, to choose and obtainappropriate human immunoglobulin genes, to graft the CDRs into thesegenes, to modify selected framework residues, to express the CDR-graftedantibody in appropriate host cells, e.g. Chinese hamster ovary (CHO)cells, and to test the resulting recombinant antibodies for bindingaffinity and specificity.

To obtain a humanized antibody, the antigen binding sites, which areformed by the CDRs of the heavy chain and CDRs of the light chain, areexcised from the DNA of cells secreting the rodent (murine) monoclonalantibody and grafted into the DNA coding for the framework of the humanantibody.

Alternatively to CDR grafting, non-human, in particular murine,anti-CD37 antibodies can be humanized by the so-called “resurfacing”technology, whereby the rodent frameworks are left unchanged with theexception of surface-exposed residues, as described in U.S. Pat. No.5,639,641.

In a further aspect, the invention relates to humanized antibodieshaving a variable heavy chain with a sequence shown in SEQ ID NO:6 and avariable light chain with a sequence selected from the sequences shownin SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20and SEQ ID NO:22.

In another aspect, the invention relates to humanized antibodies havinga variable heavy chain with a sequence shown in SEQ ID NO:8 and avariable light chain with a sequence selected from the sequences shownin SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20and SEQ ID NO:22.

In another aspect, the invention relates to humanized antibodies havinga variable heavy chain with a sequence shown in SEQ ID NO:10 and avariable light chain with a sequence selected from the sequences shownin SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20and SEQ ID NO:22.

The above defined humanized antibodies are shown in Table 1.

In certain embodiments, the humanized antibody has a human heavy chainconstant region IgG1 and a human light chain constant region kappa. Asdescribed above for the chimeric antibodies, the constant regions may beselected from other classes and subclasses.

In certain embodiments, in humanized antibodies of the invention, thehuman constant heavy chain IgG1 is an IgG1 molecule with the sequenceshown in SEQ ID NO:24 or a mutated IgG1 molecule derived therefrom andthe human light chain constant region kappa has the sequence shown inSEQ ID NO:26.

Anti-CD37 antibody molecules of the invention may also be variants ofthe antibodies that are defined by the amino acid sequences shown in thesequence listing. Using routinely available technologies, the personskilled in the art will be able to prepare, test and utilize functionalvariants of the above-defined antibodies. Examples are variantantibodies with at least one position in a CDR and/or framework altered,variant antibodies with single amino acid substitutions in the frameworkregion where there is a deviation from the germline sequence, antibodieswith conservative amino substitutions, antibodies that are encoded byDNA molecules that hybridize, under stringent conditions, with the DNAmolecules presented in the sequence listing encoding antibody variablechains.

Given the properties of individual amino acids, rational substitutionscan be performed to obtain antibody variants that conserve the overallmolecular structure of the starting antibody. Amino acid substitutions,i.e., “conservative substitutions”, may be made, for instance, on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the respective aminoacid. The skilled person is familiar with commonly practiced amino acidsubstitutions, as described e.g. in WO 2007/042309, and methods forobtaining thus modified antibodies. Given the genetic code andrecombinant and synthetic DNA techniques, DNA molecules encoding variantantibodies with one or more conservative amino acid exchanges can beroutinely designed and the respective antibodies readily obtained.

In comparison with an antibody as defined by its variable chains shownin the sequence listing, antibody variants encompassed by the inventionhave a sequence identity in the CDR regions of at least 60%, morepreferably, at least 70% or 80%, still more preferably at least 90% andmost preferably at least 95%. Preferred antibodies also have a sequencesimilarity in the CDR regions of at least 80%, more preferably 90% andmost preferably 95%. Preferred antibody variants have a sequenceidentity in the variable regions of at least 60%, more preferably, atleast 70% or 80%, still more preferably at least 90% and most preferablyat least 95%. Preferred antibodies also have a sequence similarity inthe variable regions of at least 80%, more preferably 90% and mostpreferably 95%.

“Sequence identity” between two polypeptide sequences indicates thepercentage of amino acids that are identical between the sequences.“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions.

A variant may also be obtained by using an antibody with a definedsequence as shown in the sequence listing as a starting point foroptimization and diversifying one or more amino acid residues,preferably amino acid residues in one or more CDRs, and by screening theresulting collection of antibody variants for variants with improvedproperties. Diversification of one or more amino acid residues in CDR3of the variable light chain, CDR3 of the variable heavy chain, CDR1 ofthe variable light and/or CDR2 of the variable heavy chain has beenproven useful. Diversification can be done by methods known in the art,e.g. the so-called TRIM technology referred to in WO 2007/042309.

In a further embodiment, the anti-CD37 antibody molecule of theinvention is an “affinity matured” antibody.

An “affinity matured” anti-CD37 antibody is an anti-CD37 antibodyderived from an antibody with the sequences shown in the sequencelisting, that has one or more alterations in one or more CDRs whichresult in an improvement in the affinity for the antigens, compared tothe respective original non-matured antibody. One of the procedures forgenerating such antibody mutants involves phage display (Hawkins et al.,1992; and Lowman et al., 1991). Briefly, several hypervariable regionsites (e.g. 6-7 sites) are mutated to generate all possible amino acidsubstitutions at each site. The antibody mutants thus generated aredisplayed in a monovalent fashion from filamentous phage particles asfusions to the gene III product of M13 packaged within each particle.The phage-displayed mutants are then screened for their biologicalactivity (e.g. binding affinity) as herein disclosed.

Affinity matured antibodies may also be produced by methods asdescribed, for example, by Marks et al., 1992, (affinity maturation byvariable heavy chain (VH) and variable light chain (VL) domainshuffling), or Barbas, et al., 1994; Shier et al., 1995; Yelton et al.,1995; Jackson et al., 1995; and Hawkins et al., 1992 (random mutagenesisof CDR and/or framework residues). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.In a further embodiment, the anti-CD37 antibody molecule of theinvention is a “de-immunized” antibody.

A “de-immunized” anti-CD37 antibody is an antibody derived from ahumanized or chimeric antibody with a sequence shown in the sequencelisting, that has one or more alterations in its amino acid sequencewhich result in a reduction of immunogenicity of the antibody, comparedto the respective original non-dehumanized antibody. One of theprocedures for generating such antibody mutants involves theidentification and removal of T-cell epitopes of the antibody molecule(Baker and Jones, 2007). In a first step, the immunogenicity of theantibody molecule can be determined by several methods, e.g. by in vitrodetermination of T-cell epitopes or in silico prediction of suchepitopes, as has been described in the literature (Jones et al., 2004;Jones et al., 2005; Reche et al., 2004; Hertz et al., 2006). Once thecritical residues for T-cell epitope function have been identified,mutations can be made to remove immunogenicity and retain antibodyactivity (Jones et al., 2005; Tangri et al., 2005). Methods forintroduction of mutations in proteins are well-known in the art, e.g. byoverlapping PCR techniques.

Since the Fc region of an antibody interacts with a number of Fcreceptors, which results in a number of important functionalcapabilities (which are referred to as “effector functions”), theantibody is, in certain embodiments, a full length antibody or anantibody that contains a portion of the Fc region, the latter as long asthe antibody exhibits specific binding both to the relevant portion ofthe antigen and to Fc receptors. The choice of the type and length ofthe constant region depends on whether effector functions likecomplement fixation or antibody-dependent cell-mediated cytotoxicity aredesirable features, and on the desired pharmacological properties of theantibody protein.

In an embodiment of the invention, the anti-CD37 antibody is a chimericor humanized antibody with an Fc region, or the relevant sectionthereof, that has been engineered to modulate effector functions, inparticular to enhance binding of the antibody to one or more Fcreceptors, thereby enhancing the effector function ADDC. Engineering ofthe Fc region mediates the antibody's effector function in the presenceof effector cells more effectively than that of the non-Fc-engineeredparent antibody. In one embodiment, such antibody variant mediates ADCCthat is greater than that mediated by the parent antibody. (In thefollowing, if not otherwise stated, the term “parent” in the context ofan antibody molecule, or in the context of IgG or the Fc region, refersto the non-engineered antibody molecule, Fc region or IgG, respectively,from which the mutated (engineered) molecule is derived.)

A variety of modifications of the Fc region have been suggested in theart, both in the scientific literature and in patent documents, e.g. inEP 0307434, WO 9304173, WO 9734631, WO 9744362, WO 9805787, WO 9943713,WO 9951642, WO 9958572, WO 02060919, WO 03074679, WO 2004016750, WO2004029207, WO 2004063351, WO 2004074455, WO 2004035752, WO 2004099249,WO 2005077981, WO 2005092925, WO 2006019447, WO 2006031994, WO2006047350, WO 2006053301, WO 2006088494 and WO 2007041635.

In preferred embodiments, the antibodies of the invention are Fcvariants with amino acid substitutions at positions 332 and/or 239and/or 236. In preferred embodiments, the antibodies of the inventionhave mutations in the Fc domain selected from the group of

-   -   i) a single substitution at position 332, preferably I332E;    -   ii) a combination of substitutions at positions 239 and 332,        preferably S239D/I332E;    -   iii) a combination of substitutions at positions 236 and 332,        preferably G236A/I332E;    -   iv) a combination of substitutions at positions 236, 239 and,        332, preferably G236A/S239D/I332E.

The above defined substitutions have, for example, been described byLazar et al., 2006, in WO 2004029207 and WO 2007041635.

The Fc variants in the antibodies of the present invention are definedaccording to the amino acid modifications that compose them. Thus, forexample, I332E is an Fc variant with the substitution I332E relative tothe parent Fc polypeptide. Likewise, S239D/I332E defines an Fc variantwith the substitutions S239D and I332E and S239D/I332E/G236A defines anFc variant with the substitutions S239D, I332E, and G236A relative tothe parent Fc polypeptide.

Numbering is according to the EU numbering scheme (Kabat et al., 1991),which refers to the numbering of the EU antibody (Edelman et al., 1969).The person skilled in the art will appreciate that these conventionsconsist of nonsequential numbering in specific regions of animmunoglobulin sequence, enabling a normalized reference to conservedpositions in immunoglobulin families.

In the above defined antibodies, the substituted positions 236, 239 and332 correspond to positions 119, 122 and 215, respectively, of the IgG1heavy chain depicted in SEQ ID NO:24. (In the full-length sequences ofthe heavy chains of antibodies A2, A4, B2 and B4 shown in SEQ ID NOs:28, 32, 36 and 40, the substituted amino acids are at positions 235, 238and 331).

In certain embodiments, the Fc variants of the invention are based onhuman IgG sequences, and thus human IgG sequences are used as the “base”sequences against which other sequences are compared. For the antibodiesof the present invention, the engineered Fc region is preferably IgG, inparticular IgG1, but it may also be IgG2 or variant sequences from otherimmunoglobulin classes such as IgA, IgE, IgGD, IgM or chimeric versionsof two or more immunoglobulin classes (e.g. IgG2/IgG1) and the like.Although the Fc variants of the present invention are engineered in thecontext of one parent IgG, the variants may be engineered in or“transferred” to the context of another, second parent IgG. This is doneby determining the “equivalent” or “corresponding” residues andsubstitutions between the first and second IgG, typically based onsequence or structural homology between the sequences of the first andsecond IgGs. In order to establish homology, the amino acid sequence ofa first IgG outlined herein is directly compared to the sequence of asecond IgG. After aligning the sequences, using one or more of thehomology alignment programs known in the art, allowing for necessaryinsertions and deletions in order to maintain alignment (i.e., avoidingthe elimination of conserved residues through arbitrary deletion andinsertion), the residues equivalent to particular amino acids in theprimary sequence of the first Fc variant are defined. Regardless of howequivalent or corresponding residues are determined, and regardless ofthe identity of the parent IgG in which the IgGs are made, what is meantto be conveyed is that the Fc variants used in the present invention maybe engineered into any second parent IgG that has significant sequenceor structural homology with the Fc variant. Thus for example, if avariant antibody is generated wherein the parent antibody is human IgG1,by using the methods described above or other methods for determiningequivalent residues, the variant antibody may be engineered, forexample, in a human IgG2 parent antibody, a human IgA parent antibody(see WO 2007041635).

The antibodies of the invention target the antigen CD37, which may beadvantageous over targeting CD20 in diseases in which the level of CD37expression is higher than that of CD20, as e.g. in chronic lymphocyticleukemia, where samples have shown high levels of CD37 mRNA expressioncompared to low level expression of CD20 mRNA.

It has been shown that antibodies of the invention are superior torituximab, a registered anti-CD20 antibody, with respect to ADCCactivity on Ramos cells, normal B cell depletion in whole blood andRamos Burkitt's lymphoma cell depletion. As could be shown in theexperiments of the invention, the antibodies of the invention (both thenon-Fc engineered and the Fc-engineered ones) have a B cell depletingactivity that is superior to that of rituximab. The antibodies with themutated Fc region show a ca. 10 fold increase of B cell depletionactivity as compared to rituximab (FIG. 11B).

Representatives of CD37 antibodies of the invention show potentpro-apoptotic activity without cross-linking; in this respect,antibodies with this property are superior to the anti-CD37 SMIPTru16.4, which does not show apoptosis without cross-linking (Zhao etal., 2007). Induction of apoptosis without cross-linking, which could beshown for antibodies of the invention both with and without Fcengineering, is advantageous in the absence of a cross-linking agent invivo (e.g. effector cells harboring Fcγ receptors) or at low density ofthe target antigen CD37 (e.g. tumor cells with low level expression ofCD37). An antibody which induces apoptosis without cross-linking maystill cause cell death, whereas an antibody dependent on cross-linkingdoes not.

In a further aspect, an anti-CD37 antibody molecule of the invention isan antibody fragment that is derived from a humanized or chimeric CD37specific antibody according to the present invention. To obtain antibodyfragments, e.g. Fab fragments, digestion can be accomplished by means ofroutine techniques, e.g. using papain. Examples of papain digestion aredescribed in WO 94/29348 and U.S. Pat. No. 4,342,566. Papain digestionof antibodies typically produces two identical antigen bindingfragments, so-called Fab fragments, each with a single antigen bindingsite, and a residual Fc fragment. Pepsin treatment yields an F(ab′)₂fragment that has two antigen combining sites and is still capable ofcross-linking the antigen.

The Fab fragments obtained by digestion of the antibody also contain theconstant domains of the light chain and the first constant domain (CH₁)of the heavy chain. Fab′ fragments differ from Fab fragments in thatthey contain additional residues at the carboxy terminus of the heavychain CH₁ domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragmentswhich have hinge cysteines between them. Antibody fragments can also begenerated by molecular biology methods producing the respective codingDNA fragments.

The antibody molecule will typically be a tetramer consisting of twolight chain/heavy chain pairs, but may also be dimeric, i.e. consist ofa light chain/heavy chain pair, e.g. a Fab or Fv fragment, or it may bea monomeric single chain antibody (scFv; Johnson and Bird, 1991), aminibody, or a diabody.

The anti-CD37 antibody molecule may also be in the form of a conjugate,i.e. an antibody molecule that is chemically coupled to a cytotoxicagent, particularly a cytotoxic agent that induces cytotoxicity (e.g.apoptosis or mitotic arrest) of tumor cells. As a result of normalpharmacologic clearance mechanisms, an antibody employed in a drugconjugate (an “immunoconjugate”) contacts and binds to target cells onlyin limited amounts. Therefore, the cytotoxic agent employed in theconjugate must be highly cytotoxic such that sufficient cell killingoccurs to elicit a therapeutic effect. As described in US 2004/0241174,examples of such cytotoxic agents include taxanes (see, e.g. WO 01/38318and WO 03/097625), DNA-alkylating agents (e.g., CC-1065 analogs),anthracyclines, tubulysin analogs, duocarmycin analogs, doxorubicin,auristatin E, ricin A toxin, and cytotoxic agents comprising a reactivepolyethylene glycol moiety (see, e.g., Sasse et al., 2000; Suzawa etal., 2000; Ichimura et al., 1991; Francisco et al., 2003; U.S. Pat. No.5,475,092; U.S. Pat. No. 6,340,701; U.S. Pat. No. 6,372,738; and U.S.Pat. No. 6,436,931; US 2001/0036923; US 2004/0001838; US 2003/0199519;and WO 01/49698).

In a preferred embodiment, the cytotoxic agent is a maytansinoid, i.e. aderivative of maytansine (CAS 35846538), maytansinoids being known inthe art to include maytansine, maytansinol, C-3 esters of maytansinol,and other maytansinol analogues and derivatives (see, e.g., U.S. Pat.No. 5,208,020; and U.S. Pat. No. 6,441,163).

Anti-CD37 antibody immunoconjugates may be designed and synthesized asdescribed in WO 2007/077173 for anti-FAP immunoconjugates.

In a further embodiment, the anti-CD37 molecule of the invention may beradioactively labeled to form an radioimmunoconjugate, an approachsuggested for the anti-CD37 antibody MB-1 (Buchsbaum et al., 1992, seeabove). Radionuclides with advantageous radiation properties are knownin the art, examples are Phosphorus-32, Strontium-89, Yttrium-90,Iodine-131, Samarium-153, Erbium-169, Ytterbium-175, Rhenium-188, thathave been successfully and stably coupled to MAbs. The anti-CD37antibody molecules of the invention may be labeled with variousradionuclides using direct labeling or indirect labeling methods knownin the art, as described in U.S. Pat. No. 6,241,961. A review ontechnologies for generating and applying novel radiolabeled antibodyconjugates that are useful in the present invention, is given byGoldenberg and Sharkey, 2007.

An antibody molecule of the invention, whether Fc-engineered or not, mayalso be bispecific, i.e. an antibody molecule that binds to twodifferent targets, one of them being CD37, the other one being selectedfrom e.g. surface antigens expressed by T cells, e.g. CD3, CD16 andCD56.

The present invention also relates to DNA molecules that encode thechimeric or humanized anti-CD37 antibody molecules of the invention. Thesequences encoding variable heavy chains of the antibody molecules ofthe invention are shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7 and SEQID NO:9. The sequences encoding variable light chains of the antibodymolecules of the invention are shown in SEQ ID NO:3, SEQ ID NO:11, SEQID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO: 21.

Nucleic acid molecules coding for the light chain and the heavy chainmay be synthesized chemically and enzymatically (PCR amplification) bystandard methods. First, suitable oligonucleotides can be synthesizedwith methods known in the art (e.g. Gait, 1984), which can be used toproduce a synthetic gene. Methods to generate synthetic genes fromoligonucleotides are known in the art (e.g. Stemmer et al., 1995; Ye etal., 1992; Hayden et Mandecki, 1988; Frank et al., 1987).

The DNA molecules of the invention include, but are not limited to, theDNA molecules shown in the sequence listing. Accordingly, the presentinvention also relates to nucleic acid molecules that hybridize to theDNA molecules set forth in the sequence listing under high stringencybinding and washing conditions, as defined in WO 2007/042309, where suchnucleic molecules encode an antibody or functional fragment thereof thathas properties equivalent or superior to an antibody encoded by asequence shown in the sequence listing. Preferred molecules (from anmRNA perspective) are those that have at least 75% or 80% (preferably atleast 85%, more preferably at least 90% and most preferably at least95%) homology or sequence identity with one of the DNA moleculesdescribed herein.

Yet another class of DNA variants that are within the scope of theinvention may be defined with reference to the polypeptide they encode.These DNA molecules deviate with respect to their sequence from thosedepicted in the sequence listing, but encode, due to the degeneracy ofthe genetic code, antibodies with the identical amino acid sequences. Byway of example, in view of expressing the antibodies in eukaryoticcells, the DNA sequences shown in the sequence listing have beendesigned to match codon usage in eukaryotic cells. If it is desired toexpress the antibodies in E. coli, these sequences can be changed tomatch E. coli codon usage. Variants of DNA molecules of the inventioncan be constructed in several different ways, as described e.g. in WO2007/042309.

For producing the recombinant anti-CD37 antibody molecules of theinvention, the DNA molecules encoding full-length light and heavy chainsor fragments thereof are inserted into an expression vector such thatthe sequences are operatively linked to transcriptional andtranslational control sequences.

For manufacturing the antibodies of the invention, the skilled artisanmay choose from a great variety of expression systems well known in theart, e.g. those reviewed by Kipriyanow and Le Gall, 2004.

Expression vectors include plasmids, retroviruses, cosmids, EBV-derivedepisomes, and the like. The expression vector and expression controlsequences are selected to be compatible with the host cell. The antibodylight chain gene and the antibody heavy chain gene can be inserted intoseparate vectors. In certain embodiments, both DNA sequences areinserted into the same expression vector. Convenient vectors are thosethat encode a functionally complete human CH or CL immunoglobulinsequence, with appropriate restriction sites engineered so that any VHor VL sequence can be easily inserted and expressed, as described above.The constant chain is usually kappa or lambda for the antibody lightchain, for the antibody heavy chain, it can be, without limitation, anyIgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulins, includingallelic variants.

The recombinant expression vector may also encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. The DNAencoding the antibody chain may be cloned into the vector such that thesignal peptide is linked in-frame to the amino terminus of the matureantibody chain DNA. The signal peptide may be an immunoglobulin signalpeptide or a heterologous peptide from a non-immunoglobulin protein.Alternatively, the DNA sequence encoding the antibody chain may alreadycontain a signal peptide sequence.

In addition to the DNA sequences encoding the antibody chains, therecombinant expression vectors carry regulatory sequences includingpromoters, enhancers, termination and polyadenylation signals and otherexpression control elements that control the expression of the antibodychains in a host cell. Examples for promoter sequences (exemplified forexpression in mammalian cells) are promoters and/or enhancers derivedfrom (CMV) (such as the CMV Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)), polyoma and strong mammalian promoters such as nativeimmunoglobulin and actin promoters. Examples for polyadenylation signalsare BGH polyA, SV40 late or early polyA; alternatively, 3′UTRs ofimmunoglobulin genes etc. can be used.

The recombinant expression vectors may also carry sequences thatregulate replication of the vector in host cells (e.g. origins ofreplication) and selectable marker genes. Nucleic acid moleculesencoding the heavy chain or an antigen-binding portion thereof and/orthe light chain or an antigen-binding portion thereof of an anti-CD37antibody, and vectors comprising these DNA molecules can be introducedinto host cells, e.g. bacterial cells or higher eukaryotic cells, e.g.mammalian cells, according to transfection methods well known in theart, including liposome-mediated transfection, polycation-mediatedtransfection, protoplast fusion, microinjections, calcium phosphateprecipitation, electroporation or transfer by viral vectors.

Preferably, the DNA molecules encoding the heavy chain and the lightchain are present on two vectors which are co-transfected into the hostcell, preferably a mammalian cell.

Mammalian cell lines available as hosts for expression are well known inthe art and include, inter alia, Chinese hamster ovary (CHO, CHO-DG44)cells, NSO, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human carcinoma cells (e.g., Hep G2), A549cells, 3T3 cells or the derivatives/progenies of any such cell line.Other mammalian cells, including but not limited to human, mice, rat,monkey and rodent cells lines, or other eukaryotic cells, including butnot limited to yeast, insect and plant cells, or prokaryotic cells suchas bacteria may be used. The anti-CD37 antibody molecules of theinvention are produced by culturing the host cells for a period of timesufficient to allow for expression of the antibody molecule in the hostcells.

Antibody molecules are preferably recovered from the culture medium as asecreted polypeptide or it can be recovered from host cell lysates iffor example expressed without a secretory signal. It is necessary topurify the antibody molecules using standard protein purificationmethods used for recombinant proteins and host cell proteins in a waythat substantially homogenous preparations of the antibody are obtained.By way of example, state-of-the art purification methods useful forobtaining the anti-CD37 antibody molecule of the invention include, as afirst step, removal of cells and/or particulate cell debris from theculture medium or lysate. The antibody is then purified from contaminantsoluble proteins, polypeptides and nucleic acids, for example, byfractionation on immunoaffinity or ion-exchange columns, ethanolprecipitation, reverse phase HPLC, Sephadex chromatography,chromatography on silica or on a cation exchange resin. As a final stepin the process for obtaining an anti-CD37 antibody molecule preparation,the purified antibody molecule may be dried, e.g. lyophilized, asdescribed below for therapeutic applications.

In a further aspect, the present invention relates to a pharmaceuticalcomposition containing, as the active ingredient, the anti-CD37 antibodymolecule of the invention.

To be used in therapy, the anti-CD37 antibody is included intopharmaceutical compositions appropriate to facilitate administration toanimals or humans. Typical formulations of the anti-CD37 antibodymolecule can be prepared by mixing the anti-CD37 antibody molecule withphysiologically acceptable carriers, excipients or stabilizers, in theform of lyophilized or otherwise dried formulations or aqueous solutionsor aqueous or non-aqueous suspensions. Carriers, excipients, modifiersor stabilizers are nontoxic at the dosages and concentrations employed.They include buffer systems such as phosphate, citrate, acetate andother anorganic or organic acids and their salts; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone or polyethylene glycol (PEG); amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, oligosaccharides or polysaccharides andother carbohydrates including glucose, mannose, sucrose, trehalose,dextrins or dextrans; chelating agents such as EDTA; sugar alcohols suchas, mannitol or sorbitol; salt-forming counter-ions such as sodium;metal complexes (e.g. Zn-protein complexes); and/or ionic or non-ionicsurfactants such as TWEEN™ (polysorbates), PLURONICS™ or fatty acidesters, fatty acid ethers or sugar esters. Also organic solvents can becontained in the antibody formulation such as ethanol or isopropanol.The excipients may also have a release-modifying or absorption-modifyingfunction.

The anti-CD37 antibody molecules may also be dried (freeze-dried,spray-dried, spray-freeze dried, dried by near or supercritical gases,vacuum dried, air-dried), precipitated or crystallized or entrapped inmicrocapsules that are prepared, for example, by coacervation techniquesor by interfacial polymerization using, for example,hydroxymethylcellulose or gelatin and poly-(methylmethacylate),respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), in macroemulsions or precipitated or immobilized ontocarriers or surfaces, for example by pcmc technology (protein coatedmicrocrystals). Such techniques are disclosed in Remington: The Scienceand Practice of Pharmacy, 21^(st) edition, Hendrickson R. Ed.

Naturally, the formulations to be used for in vivo administration mustbe sterile; sterilization may be accomplished be conventionaltechniques, e.g. by filtration through sterile filtration membranes.

It may be useful to increase the concentration of the anti-CD37 antibodyto come to a so-called high concentration liquid formulation (HCLF);various ways to generate such HCLFs have been described.

The anti-CD37 antibody molecule may also be contained in asustained-release preparation. Such preparations include solid,semi-solid or liquid matrices of hydrophobic or hydrophilic polymers,and may be in the form of shaped articles, e.g. films, sticks ormicrocapsules and may be applied via an application device. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate) or sucrose acetate butyrate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilization (e.g. asdescribed in WO 89/011297) from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

Formulations that may also be used for the anti-CD37 antibody moleculeof the invention are described in U.S. Pat. No. 7,060,268 and U.S. Pat.No. 6,991,790.

The CD37 antibody molecule can be incorporated also in other applicationforms, such as dispersions, suspensions or liposomes, tablets, capsules,powders, sprays, transdermal or intradermal patches or creams with orwithout permeation enhancing devices, wafers, nasal, buccal or pulmonaryformulations, or may be produced by implanted cells or—after genetherapy—by the individual's own cells.

An anti-CD37 antibody molecule may also be derivatized with a chemicalgroup such as polyethylene glycol (PEG), a methyl or ethyl group, or acarbohydrate group. These groups may be useful to improve the biologicalcharacteristics of the antibody, e.g. to increase serum half-life or toincrease tissue binding.

The preferred mode of application is parenteral, by infusion orinjection (intravenous, intramuscular, subcutaneous, intraperitoneal,intradermal), but other modes of application such as by inhalation,transdermal, intranasal, buccal, oral, may also be applicable.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, the severityand course of the disease, whether the antibody is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 0.01 μg/kg to40 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays, e.g. by determining the extent of B cell depletion (e.g.using flow cytometry).

The “therapeutically effective amount” of the antibody to beadministered is the minimum amount necessary to prevent, ameliorate, ortreat a disease or disorder.

The anti-CD37 antibody molecule of the invention and pharmaceuticalcompositions containing it are useful to deplete B cells that expressCD37 on their surface and that cause cancerous orautoimmune/inflammatory disease.

In a first aspect, the pharmaceutical composition of the invention isuseful for the treatment of cancers, in particular any CD37-positivemalignancies.

B cell malignancies include, without limitation, B cell lymphomas (e.g.various forms of Hodgkin's disease, B cell non-Hodgkin's lymphoma (NHL)and related lymphomas (e.g. Waldenstrom's macroglobulinaemia (alsocalled lymphoplasmacytic lymphoma or immunocytoma) or central nervoussystem lymphomas), leukemias (e.g. acute lymphoblastic leukemia (ALL),chronic lymphocytic leukemia (CLL; also termed B cell chroniclymphocytic leukemia BCLL), hairy cell leukemia and chronic myoblasticleukemia) and myelomas (e.g. multiple myeloma). Additional B cellmalignancies include small lymphocytic lymphoma, B cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, solitary plasmacytoma of bone, extraosseousplasmacytoma, extra-nodal marginal zone B cell lymphoma ofmucosa-associated (MALT) lymphoid tissue, nodal marginal zone B celllymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large Bcell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascularlarge B cell lymphoma, primary effusion lymphoma, Burkitt'slymphoma/leukemia, grey zone lymphoma, B cell proliferations ofuncertain malignant potential, lymphomatoid granulomatosis, andpost-transplant lymphoproliferative disorder.

In a further aspect, a pharmaceutical composition containing anti-CD37antibodies is useful for the treatment of autoimmune and inflammatorydiseases that involve B cells in their pathology.

Such diseases include, but are not limited to: arthritis, rheumatoidarthritis, juvenile rheumatoid arthritis, osteoarthritis,polychondritis, psoriatic arthritis, psoriasis, dermatitis,polymyositis/dermatomyositis, inclusion body myositis, inflammatorymyositis, toxic epidermal necrolysis, systemic scleroderma andsclerosis, CREST syndrome, responses associated with inflammatory boweldisease, Crohn's disease, ulcerative colitis, respiratory distresssyndrome, adult respiratory distress syndrome (ARDS), meningitis,encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions,eczema, asthma, conditions involving infiltration of T cells and chronicinflammatory responses, atherosclerosis, autoimmune myocarditis,leukocyte adhesion deficiency, systemic lupus erythematosus (SLE),subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis,lupus cerebritis, juvenile onset diabetes, multiple sclerosis, allergicencephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham'schorea, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis,sarcoidosis, granulomatosis including Wegener's granulomatosis andChurg-Strauss disease, agranulocytosis, vasculitis (includinghypersensitivity vasculitis/angiitis, ANCA and rheumatoid vasculitis),aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia, purered cell aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia, pancytopenia, leukopenia, diseases involvingleukocyte diapedesis, central nervous system (CNS) inflammatorydisorders, multiple organ injury syndrome, myasthenia gravis,antigen-antibody complex mediated diseases, anti-glomerular basementmembrane disease, anti-phospho lipid antibody syndrome, allergicneuritis, Behcet disease, Castleman's syndrome, Goodpasture's syndrome,Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen'ssyndrome, Stevens-Johnson syndrome, solid organ transplant rejection,graft versus host disease (GVHD), pemphigoid bullous, pemphigus,autoimmune polyendocrinopathies, seronegative spondyloarthropathies,Reiter's disease, stiff-man syndrome, giant cell arteritis, immunecomplex nephritis, IgA nephropathy, IgM polyneuropathies or IgM mediatedneuropathy, idiopathic thrombocytopenic purpura (ITP), thromboticthrombocytopenic purpura (TTP), Henoch-Schonlein purpura, autoimmunethrombocytopenia, autoimmune disease of the testis and ovary includingautoimmune orchitis and oophoritis, primary hypothyroidism; autoimmuneendocrine diseases including autoimmune thyroiditis, chronic thyroiditis(Hashimoto's Thyroiditis), subacute thyroiditis, idiopathichypothyroidism, Addison's disease, Grave's disease, autoimmunepolyglandular syndromes (or polyglandular endocrinopathy syndromes),Type I diabetes also referred to as insulin-dependent diabetes mellitus(IDDM) and Sheehan's syndrome; autoimmune hepatitis, lymphoidinterstitial pneumonitis (HIV), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barre' Syndrome, large vesselvasculitis (including polymyalgia rheumatica and giant cell (Takayasu's)arteritis), medium vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa), polyarteritis nodosa (PAN) ankylosingspondylitis, Berger's disease (IgA nephropathy), rapidly progressiveglomerulonephritis, primary biliary cirrhosis, Celiac sprue (glutenenteropathy), cryoglobulinemia, cryoglobulinemia associated withhepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease,familial Mediterranean fever, microscopic polyangiitis, Cogan'ssyndrome, Whiskott-Aldrich syndrome and thromboangiitis obliterans (seeWO 2007/014278).

Depending on the disorder to be treated, the anti-CD37 antibody moleculeof the invention may be used on its own or in combination with one ormore additional therapeutic agents, in particular selected from DNAdamaging or tubulin binding agents or therapeutically active compoundsthat inhibit angiogenesis, signal transduction pathways or mitoticcheckpoints in cancer cells.

The additional therapeutic agent may be administered simultaneouslywith, optionally as a component of the same pharmaceutical preparation,or before or after administration of the anti-CD37 antibody molecule.

In certain embodiments, the additional therapeutic agent may be, withoutlimitation, one or more inhibitors selected from the group of inhibitorsof EGFR family, VEGFR family, IGF-1R, Insulin receptors, AuroraA,AuroraB, PLK and PI3 kinase, FGFR, PDGFR, Raf, KSP or PDK1.

Further examples of additional therapeutic agents are inhibitors ofCDKs, Akt, Src, Bcr-Abl, cKit, cMet/HGF, c-Myc, Flt3, HSP90, hedgehogantagonists, inhibitors of JAK/STAT, Mek, mTor, NFkappaB, theproteasome, Rho, an inhibitor of Wnt signaling or Notch signaling or anubiquitination pathway inhibitor.

Examples for Aurora inhibitors are, without limitation, PHA-739358,AZD-1152, AT-9283, CYC-116, R-763, VX-667, MLN-8045, PF-3814735,SNS-314, VX-689, GSK-1070916, TTP-607, PHA-680626, MLN-8237 andENMD-2076.

An example for a PLK inhibitor is GSK-461364.

Examples for raf inhibitors are BAY-73-4506 (also a VEGFR inhibitor),PLX-4032, RAF-265 (also a VEGFR inhibitor), sorafenib (also a VEGFRinhibitor), XL-281, and Nevavar (also an inhibitor of the VEGFR).

Examples for KSP inhibitors are ispinesib, ARRY-520, AZD-4877,CK-1122697, GSK-246053A, GSK-923295, MK-0731, SB-743921, LY-2523355, andEMD-534085.

Examples for a src and/or bcr-abl inhibitors are dasatinib, AZD-0530,bosutinib, XL-228 (also an IGF-1R inhibitor), nilotinib (also a PDGFRand cKit inhibitor), imatinib (also a cKit inhibitor), NS-187, KX2-391,AP-24534 (also an inhibitor of EGFR, FGFR, Tie2, Flt3), KM-80 and LS-104(also an inhibitor of Flt3, Jak2).

An example for a PDK1 inhibitor is AR-12.

An example for a Rho inhibitor is BA-210.

Examples for PI3 kinase inhibitors are PX-866, PX-867, BEZ-235 (also anmTor inhibitor), XL-147, and XL-765 (also an mTor inhibitor), BGT-226,CDC-0941.

Examples for inhibitors of cMet or HGF are XL-184 (also an inhibitor ofVEGFR, cKit, Flt3), PF-2341066, MK-2461, XL-880 (also an inhibitor ofVEGFR), MGCD-265 (also an inhibitor of VEGFR, Ron, Tie2), SU-11274,PHA-665752, AMG-102, AV-299, ARQ-197, MetMAb, CGEN-241, BMS-777607,JNJ-38877605, PF-4217903, SGX-126, CEP-17940, AMG-458, INCB-028060, andE-7050.

An example for a c-Myc inhibitor is CX-3543.

Examples for Flt3 inhibitors are AC-220 (also an inhibitor of cKit andPDGFR), KW-2449, LS-104 (also an inhibitor of bcr-abl and Jak2),MC-2002, SB-1317, lestaurtinib (also an inhibitor of VEGFR, PDGFR, PKC),TG-101348 (also an inhibitor of JAK2), XL-999 (also an inhibitor ofcKit, FGFR, PDGFR and VEGFR), sunitinib (also an inhibitor of PDGFR,VEGFR and cKit), and tandutinib (also an inhibitor of PDGFR, and cKit).

Examples for HSP90 inhibitors are, tanespimycin, alvespimycin, IPI-504,STA-9090, MEDI-561, AUY-922, CNF-2024, and SNX-5422.

Examples for JAK/STAT inhibitors are CYT-997 (also interacting withtubulin), TG-101348 (also an inhibitor of Flt3), and XL-019.

Examples for Mek inhibitors are ARRY-142886, AS-703026, PD-325901,AZD-8330, ARRY-704, RDEA-119, and XL-518.

Examples for mTor inhibitors are temsirolimus, deforolimus (which alsoacts as a VEGF inhibitor), everolimus (a VEGF inhibitor in addition).XL-765 (also a PI3 kinase inhibitor), and BEZ-235 (also a PI3 kinaseinhibitor).

Examples for Aid inhibitors are perifosine, GSK-690693, RX-0201, andtriciribine.

Examples for cKit inhibitors are masitinib, OSI-930 (also acts as aVEGFR inhibitor), AC-220 (also an inhibitor of Flt3 and PDGFR),tandutinib (also an inhibitor of Flt3 and PDGFR), axitinib (also aninhibitor of VEGFR and PDGFR), sunitinib (also an inhibitor of Flt3,PDGFR, VEGFR), and XL-820 (also acts as a VEGFR- and PDGFR inhibitor),imatinib (also a bcr-abl inhibitor), nilotinib (also an inhibitor ofbcr-abl and PDGFR).

Examples for hedgehog antagonists are IPI-609, CUR-61414, GDC-0449,IPI-926, and XL-139.

Examples for CDK inhibitors are seliciclib, AT-7519, P-276, ZK-CDK (alsoinhibiting VEGFR2 and PDGFR), PD-332991, R-547, SNS-032, PHA-690509,PHA-848125, and SCH-727965.

Examples for proteasome inhibitors are bortezomib, carfilzomib, andNPI-0052 (also an inhibitor of NFkappaB).

Examples for proteasome inhibitors/NFkappaB pathway inhibitors arebortezomib, carfilzomib, NPI-0052, CEP-18770, MLN-2238, PR-047, PR-957,AVE-8680, and SPC-839.

An example for an inhibitor of the ubiquitination pathway is HBX-41108.

Examples for anti-angiogenic agents are inhibitors of the FGFR, PDGFRand VEGF(R), and thalidomides, such agents being selected from, withoutlimitation, bevacizumab, motesanib, CDP-791, SU-14813, telatinib,KRN-951, ZK-CDK (also an inhibitor of CDK), ABT-869, BMS-690514,RAF-265, IMC-KDR, IMC-18F1, IMiDs, thalidomide, CC-4047, lenalidomide,ENMD-0995, IMC-D11, Ki-23057, brivanib, cediranib, 1B3, CP-868596,IMC-3G3, R-1530 (also an inhibitor of Flt3), sunitinib (also aninhibitor of cKit and Flt3), axitinib (also an inhibitor of cKit),lestaurtinib (also an inhibitor of Flt3 and PKC), vatalanib, tandutinib(also an inhibitor of Flt3 and cKit), pazopanib, PF-337210, aflibercept,E-7080, CHIR-258, sorafenib tosylate (also an inhibitor of Raf),vandetanib, CP-547632, OSI-930, AEE-788 (also an inhibitor of EGFR andHer2), BAY-57-9352 (also an inhibitor of Raf), BAY-73-4506 (also aninhibitor of Raf), XL-880 (also an inhibitor of cMet), XL-647 (also aninhibitor of EGFR and EphB4), XL-820 (also an inhibitor of cKit),nilotinib (also an inhibitor of cKit and brc-abl), CYT-116, PTC-299,BMS-584622, CEP-11981, dovitinib, CY-2401401, and ENMD-2976.

The additional therapeutic agent may also be selected from EGFRinhibitors, it may be a small molecule EGFR inhibitor or an anti-EGFRantibody. Examples for anti-EGFR antibodies, without limitation, arecetuximab, panitumumab, nimotuzumab, zalutumumab; examples for smallmolecule EGFR inhibitors are gefitinib, erlotinib and vandetanib (alsoan inhibitor of the VEGFR). Another example for an EGFR modulator is theEGF fusion toxin.

Further EGFR and/or Her2 inhibitors useful for combination with ananti-CD37 antibody molecule of the invention are lapatinib, trastuzumab,pertuzumab, XL-647, neratinib, BMS-599626 ARRY-334543, AV-412, mAB-806,BMS-690514, JNJ-26483327, AEE-788 (also an inhibitor of VEGFR),AZD-8931, ARRY-380 ARRY-333786, IMC-11F8, Zemab, TAK-285, AZD-4769.

The additional drug may also be selected from agents that target theIGF-1R and insulin receptor pathways. Such agents include antibodiesthat bind to IGF-1R (e.g. CP-751871, AMG-479, IMC-A12, MK-0646,AVE-1642, R-1507, BIIB-022, SCH-717454, rhu Mab IGFR and novel chemicalentities that target the kinase domain of the IGF1-R (e.g. OSI-906 orBMS-554417, XL-228, BMS-754807).

Other agents that may be advantageously combined in a therapy with theanti-CD37 antibody molecule of the invention are molecules targetingCD20, including CD20 specific antibodies like rituximab, LY-2469298,ocrelizumab, MEDI-552, IMMU-106, GA-101 (=R7159), XmAb-0367, ofatumumab,radiolabbeled CD20 antibodies, like tositumumab and ibritumomab tiuxetanor other CD20 directed proteins, like the SMIP Tru015, PRO-131921,FBT-A05, veltuzumab, R-7159.

CD37 antibodies may be combined with inhibitors of other surfaceantigens expressed on leukocytes, in particular antibodies orantibody-like molecules, e.g. anti-CD2 (siplizumab), anti-CD4(zanolimumab), anti-CD19 (MT-103, MDX-1342, SAR-3419, XmAb-5574),anti-CD22 (epratuzumab), anti-CD23 (lumiliximab), anti-CD30(iratumumab), anti-CD32B (MGA-321), anti-CD38 (HuMax-CD38), anti-CD40(SGN40), anti-CD52 (alemtuzumab), anti-CD80 (galiximab). An antibody ofthe invention may also be combined with another CD37 antagonist, e.g.TRU-016.

Other agents to be combined with CD37 antibodies are immunotoxins likeBL-22 (an anti-CD22 immunotoxin), inotuzumab ozogamicin (an anti-CD23antibody-calicheamicin conjugate), RFT5.dgA (anti-CD25 Ricin toxinA-chain), SGN-35 (an anti-CD30-auristatin E conjugate), and gemtuzumabozogamicin (an anti-CD33 calicheamicin conjugate), MDX-1411 (anti-CD70conjugate), or radiolabelled antibodies like ⁹⁰Y-epratuzumab (anti-CD22radioimmunoconjugate).

In addition, anti-CD37 antibodies may be combined with immunomodulators,agents, e.g. antibodies, that induce apoptosis or modify signaltransduction pathways like the TRAIL receptor modulators mapatumumab (aTRAIL-1 receptor agonist), lexatumumab (a TRAIL-2 receptor agonist),tigatuzumab, Apomab, AMG-951 and AMG-655; an anti-HLA-DR antibody (like1D09C3), an anti-CD74, an osteoclast differentiation factor ligandinhibitor (like denosumab), a BAFF antagonist (like AMG-623a) or anagonist of a Toll-like receptor (e.g. TLR-4 or TLR-9).

Other drugs that may be used in combination with the anti-CD37 antibodymolecules of the present invention are selected from, but not limited tohormones, hormonal analogues and antihormonals (e.g. tamoxifen,toremifene, raloxifene, fulvestrant, megestrol acetate, flutamide,nilutamide, bicalutamide, cyproterone acetate, finasteride, buserelinacetate, fludrocortinsone, fluoxymesterone, medroxyprogesterone,hydroxyprogesterone caproate, diethylstilbestrol, testosteronepropionate, fluoxymesterone/equivalents, octreotide, arzoxifene,pasireotide, vapreotide, adrenocorticosteroids/antagonists, prednisone,dexamethasone, ainoglutethimide), aromatase inhibitors (e.g.anastrozole, letrozole, liarozole, exemestane, atamestane, formestane),LHRH agonists and antagonists (e.g. goserelin acetate, leuprolide,abarelix, cetrorelix, deslorelin, histrelin, triptorelin),antimetabolites (e.g. antifolates like methotrexate, trimetrexate,pemetrexed, pyrimidine analogues like 5-fluorouracil,fluorodeoxyuridine, capecitabine, decitabine, nelarabine, 5-azacytidine,and gemcitabine, purine and adenosine analogues such as mercaptopurine,thioguanine, azathioprine, cladribine and pentostatin, cytarabine,fludarabine, clofarabine); antitumor antibiotics (e.g. anthracyclineslike doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C,bleomycin dactinomycin, plicamycin, splicamycin, actimomycin D,mitoxantrone, mitoxantroneidarubicin, pixantrone, streptozocin,aphidicolin); platinum derivatives (e.g. cisplatin, oxaliplatin,carboplatin, lobaplatin, satraplatin); alkylating agents (e.g.estramustine, semustine, mechlorethamine, melphalan, chlorambucil,busulphan, dacarbazine, cyclophosphamide, ifosfamide, hydroxyurea,temozolomide, nitrosoureas such as carmustine and lomustine, thiotepa);antimitotic agents (e.g. vinca alkaloids like vinblastine, vindesine,vinorelbine, vinflunine and vincristine; and taxanes like paclitaxel,docetaxel and their formulations, larotaxel; simotaxel, and epothiloneslike ixabepilone, patupilone, ZK-EPO); topoisomerase inhibitors (e.g.epipodophyllotoxins like etoposide and etopophos, teniposide, amsacrine,topotecan, irinotecan, banoxantrone, camptothecin) and miscellaneouschemotherapeutics such as retinoic acid derivatives, amifostine,anagrelide, interferon alpha, interferon beta, interferon gamma,interleukin-2, procarbazine, N-methylhydrazine, mitotane, and porfimer,bexarotene, celecoxib, ethylenemine/methyl-melamine,thriethyienemelamine, triethylene thiophosphoramide, hexamethylmelamine,and enzymes L-asparaginase, L-arginase and metronidazole, misonidazole,desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, RSU 1069,EO9, RB 6145, SR4233, nicotinamide, 5-bromodeozyuridine,5-iododeoxyuridine, bromodeoxycytidine, erythrohydroxynonyl-adenine,anthracenedione, GRN-163L (a competitive telomerase templateantagonist), SDX-101 (a PPAR agonist), talabostat (a DPP inhibitor),forodesine (a PNP inhibitor), atacicept (a soluble receptor targetingTNF family members BLyS and APRIL), TNF-alpha neutralizing agents(Enbrel, Humira, Remicade), XL-844 (a CHK1/2 inhibitor), VNP-40101M (aDNA alkylating agent), SPC-2996 (an antisense bcl2 inhibitor), obatoclax(a bcl2 inhibitor), enzastaurin (a PKC beta modulator), vorinistat (anHDAC inhibitor), romidepsin (an HDAC inhibitor), AT-101 (a Bcl-2/Bcl-xLinhibitor), plitidepsin (a multi-actioned depsipeptide), SL-11047 (apolyamine metabolism modulators).

In certain embodiments, the anti-CD37 antibody molecule is appliedtogether with “CHOP” (a combination of cyclophosphamide, doxorubicin,vincristine and prednisone).

The anti-CD37 antibody molecule of the invention may also be used incombination with other therapies including surgery, radiotherapy,endocrine therapy, biologic response modifiers, hyperthermia andcryotherapy and agents to attenuate any adverse effect (e.g.antiemetics), G-CSF, GM-CSF, photosensitizers such as hematoporphyrinderivatives, Photofrin®, benzoporphyrin derivatives, Npe6, tinetioporphyrin, pheoboride-a bacteriochlorophyll-a, naphthalocyanines,phthalocyanines, zinc phthalocyanines.

Monoclonal antibodies show exquisite antigen specificity and frequentlyonly react with the human target antigen, but not with homologueproteins from animal species. To support development of therapeuticantibodies, appropriate animal models for assessment of in vivo toxicityand pharmacodynamic behavior are desirable. One possibility for an invivo model is a transgenic mouse in which the endogenous target antigenis replaced by its human homologue (“knock-out/knock-in mouse”). Inparticular, for developing therapeutic anti-CD37 antibodies, the murineCD37 gene can be replaced by the human CD37 gene. This can be achievedby constructing a targeting vector which contains the coding genomicsequence of the human CD37 gene flanked by non-translated sequences.This targeting vector can be used for homologous recombination usingmouse ES cells. Transgenic animals homozygous for human CD37 expressioncan be used to assess the pharmacodynamic effect of antibodies directedagainst human CD37, e.g. by monitoring the number of peripheral B cellsafter application of the antibodies. Alternatively, those mice can beused to investigate potential toxic effects of human CD37 specificantibodies, after i.v. application.

Another possibility in the case of lack of animal cross-reactivity ofmonoclonal antibodies is the generation of a so-called surrogateantibody. A surrogate antibody is an antibody which reacts with thehomologous protein of an animal species which is relevant and useful forinvestigation of pharmacodynamic and toxic effects, e.g. the mouse orthe cynomolgus monkey. In case of CD37, monoclonal antibodies aredeveloped which are specific for macaque CD37 or mouse CD37,respectively Ideally, such a surrogate antibody should have similarbinding and functional properties as the development antibody. This canbe investigated by the use of assay systems which utilize macaque ormouse CD37 expressing cells as target cells, e.g. for binding, FACSScatchard analysis, ADCC and apoptosis assays. Ultimately, the surrogateantibody can be selected by virtue of its B cell depleting activity inmacaque or mouse blood in vitro.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Chimeric Antibody A0 specifically recognizes the CD37 antigen,determined by FACS competition assay

FIG. 2: Binding of humanized versions of A0 to cellular CD37 antigen,determined by FACS

FIG. 3: Binding of humanized versions of A0 to cellular CD37 antigen,determined by FACS

FIG. 4: Affinity of humanized versions of A0 to cellular CD37 antigen,determined by FACS scatchard analysis

FIG. 5: ADCC activity of humanized versions of A0 on Ramos cells

FIG. 6: Pro-apoptotic activity of humanized versions of A0 on Ramoscells

FIG. 7: ADCC activity of Fc-engineered versions of mAb A0 on Ramos cells

FIG. 8: ADCC activity of Fc-engineered versions of mAb B0 on Ramos cells

FIG. 9: Pro-apoptotic activity of mAb A0 and B0

FIG. 10: Pro-apoptotic activity of Fc-engineered versions of mAb A0

FIG. 11 A: Depletion of normal human B cells in a whole blood assay byFc-engineered antibodies A2 and B2 in comparison to Rituximab

FIG. 11 B: Superior B cell depleting activity of antibodies afterFc-engineering in comparison to Rituximab

FIG. 11 C: Antibodies A2 and B2 do not deplete T cells and monocytes inwhole blood assays

FIG. 12: Superior ADCC activity after Fc-engineering compared toRituximab

FIG. 13: Depletion of Ramos Burkitt's lymphoma cells in a whole bloodassay by Fc-engineered antibodies A2 and B2 in comparison to Rituximab

FIG. 14: In vivo tumor growth inhibition of Ramos xenograft tumors innude mice by Fc-engineered antibodies A2 and B2

FIG. 15: Expression of CD37 on multiple myeloma cells

FIG. 16: ADCC activity of antibodies A2 and B2 on multiple myeloma cells

FIG. 17: Pro-apoptotic activity of antibodies A2 and B2 onpatient-derived CLL cells

EXAMPLE 1 Generation of Chimeric and Humanized Anti-CD37 Antibodies a)Generation of Chimeric Antibody A0

Based on the variable heavy and light chain amino acid sequences shownin SEQ ID NO:2 and SEQ ID NO:4, the corresponding DNA sequences aresynthesized applying codon usage optimized for mammalian cells (GeneArt,Regensburg, Germany), adding at the 5′ end a HindIII and at the 3′ end aBamH1 cloning site. The synthesized DNA molecules are digested withHindIII and BamHI and the resulting DNA fragments (SEQ ID NO:1 and SEQID NO:3 plus restriction sites) are cloned into pcDNA3.1 basedexpression vectors coding for human IgG1 constant region and human kappalight chain constant region, respectively (SEQ ID NO:24 and SEQ IDNO:26). EndoFree plasmid preparations (Qiagen) are prepared and theheavy and light chain plasmids are co-transfected into HEK293 freestylecells (Invitrogen) at a concentration of 1 mg/L of each plasmidaccording to the supplier's protocol. After 72 hours the supernatant isharvested and the IgG concentration is determined by ELISA. Theresulting chimeric anti-CD37 antibody (designated A0) is purified on amodified protein A column (GE Healthcare), eluted into a citrate bufferthen dialyzed in PBS.

b) Generation of Humanized Versions of Chimeric Antibody A0

Humanization of chimeric mAb A0, as obtained in a), is performed using aCDR grafting approach, as described e.g. in U.S. Pat. No. 5,225,539;U.S. Pat. No. 6,548,640; U.S. Pat. No. 6,982,321.

To establish a structural model of the mAb A0 VL domain, a structuraltemplate is chosen from the Protein Data Bank (PDB) of BrookhavenNational Laboratory. The VL domain from the murine monoclonal antibodyentry “1KB5” is chosen with 88% sequence identity/81% similarity and 2.5Å resolution. For the mAb A0 VH domain, the same mouse monoclonalantibody structure “1KB5” with 90% sequence identity and 91% similarityis chosen as the main modeling template. The best fit for humanconsensus framework is found to be of the type human Vkappa1 (hVK1) andhuman VH1 (hVH1). As an alternative design, a graft to the most stablehuman consensus domains hVK3 and hVH3 is chosen. For grafting, the mAbA0_VL and mAb A0_VH models are combined with human consensus domainmodels hVK1, hVK3, hVH1A and hVH3 and combined to produce to Fv-models.Loop grafting is performed by embedding the murine mAb A0 CDR regionsinto the human antibody frameworks and the DNA molecules of thehumanized chain constructs are synthesized.

The respective humanized variable regions are synthesized and clonedinto immunoglobulin expression vectors and transiently expressed in theHEK 293 freestyle expression system (Invitrogen), as described in a), inthe combinations of heavy and light chain sequences as shown in Table 1,and purified on protein A columns.

TABLE 1 Sequences of heavy and light variable chains of the chimeric andhumanized anti-CD37 antibodies used in the Examples. Heavy Chain LightChain SEQ ID NO: SEQ ID NO: Antibody aa/DNA aa/DNA A (=A0) seq 2/1 seq4/3 B seq 6/5 seq 12/11 C seq 6/5 seq 14/13 D seq 6/5 seq 16/15 H seq8/7 seq 18/17 I seq 8/7 seq 20/19 J seq 8/7 seq 22/21 K seq 10/9 seq18/17 L seq 10/9 seq 20/19 M seq 10/9 seq 22/21

c) Generation of Fc-Engineered Chimeric and Humanized Anti-CD37Antibodies

The generation of Fc mutants is performed as described by Lazar et al.,2006. The resulting Fc-engineered heavy chain sequence is introducedinto the expression vector pAD-CMV1 (described in EP 393 438) andco-transfected together with a plasmid containing the light chainencoding sequence into CHO-DG44 cells. The antibody is harvested fromcell culture media 5 to 7 days after transfection and purified viaprotein A chromatography, eluted into a citrate buffer then dialyzed inPBS. The protein content of the samples is determined via Protein AHPLC, the endotoxin content is determined via Kinetic-QCL KineticChromogenic Assay (Lonza). The monomer content of the samples isdetermined by HP-SEC, all samples used for functional testing show amonomer content of >95%.

TABLE 2 Sequences of heavy and light variable chains (columns III andIV) and Fc mutations (coulum II) of the chimeric and humanized anti-CD37antibodies (antibody A0, B0, C0 etc. is identical with antibody A and B,C etc. in Table 1). III IV V VI II SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQID NO: I Fc substitution(s) v heavy v light complete heavy completelight Ab (Kabat numbering) chain aa/DNA chain aa/DNA chain aa/DNA chainaa/DNA A0 — seq 2/1 seq 4/3 2/1 fused 4/3 fused to 24/23 to 26/25 A1I332E seq 2/1 seq 4/3 2/1 fused 4/3 fused to 24*/23* to 26/25 A2S239D/I332E seq 2/1 seq 4/3 28/27 30/29 A3 I332E/G236A seq 2/1 seq 4/32/1 fused 4/3 fused to 24*/23* to 26/25 A4 S239D/I332E/G236A seq 2/1 seq4/3 32/31 34/33 B0 — seq 6/5 seq 12/11 6/5 fused 12/11 fused to 24/23 to26/25 B1 I332E seq 6/5 seq 12/11 6/5 fused 12/11 fused to 24*/23* to26/25 B2 S239D/I332E seq 6/5 seq 12/11 36/35 38/37 B3 I332E/G236A seq6/5 seq 12/11 6/5 fused 12/11 fused to 24*/23* to 26/25 B4S239D/I332E/G236A seq 6/5 seq 12/11 40/39 42/41 C0 — seq 6/5 seq 14/136/5 fused 14/13 fused to 24/23 to 26/25 C1 I332E seq 6/5 seq 14/13 6/5fused 14/13 fused to 24*/23* to 26/25 C2 S239D/I332E seq 6/5 seq 14/136/5 fused 14/13 fused to 24*/23* to 26/25 C3 I332E/G236A seq 6/5 seq14/13 6/5 fused 14/13 fused to 24*/23* to 26/25 C4 S239D/I332E/G236A seq6/5 seq 14/13 6/5 fused 14/13 fused to 24*/23* to 26/25 D0 — seq 6/5 seq16/15 6/5 fused 16/15 fused to 24/23 to 26/25 D1 I332E seq 6/5 seq 16/156/5 fused 16/15 fused to 24*/23* to 26/25 D2 S239D/I332E seq 6/5 seq16/15 6/5 fused 16/15 fused to 24*/23* to 26/25 D3 I332E/G236A seq 6/5seq 16/15 6/5 fused 16/15 fused to 24/*23* to 26/25 D4 S239D/I332E/G236Aseq 6/5 seq 16/15 6/5 fused 16/15 fused to 24/*23* to 26/25 H0 — seq 8/7seq 18/17 8/7 fused 18/17 fused to 24/23 to 26/25 H1 I332E seq 8/7 seq18/17 8/7 fused 18/17 fused to 24*/23* to 26/25 H2 S239D/I332E seq 8/7seq 18/17 8/7 fused 18/17 fused to 24*/23* to 26/25 H3 I332E/G236A seq8/7 seq 18/17 8/7 fused 18/17 fused to 24*/23* to 26/25 H4S239D/I332E/G236A seq 8/7 seq 18/17 8/7 fused 18/17 fused to 24*/23* to26/25 I-0 — seq 8/7 seq 20/19 8/7 fused 20/19 fused to 24/23 to 26/25I-1 I332E seq 8/7 seq 20/19 8/7 fused 20/19 fused to 24*/23* to 26/25I-2 S239D/I332E seq 8/7 seq 20/19 8/7 fused 20/19 fused to 24*/23* to26/25 I-3 I332E/G236A seq 8/7 seq 20/19 8/7 fused 20/19 fused to 24*/23*to 26/25 I-4 S239D/I332E/G236A seq 8/7 seq 20/19 8/7 fused 20/19 fusedto 24*/23* to 26/25 J0 — seq 8/7 seq 22/21 8/7 fused 22/21 fused to24/23 to 26/25 J1 I332E seq 8/7 seq 22/21 8/7 fused 22/21 fused to24*/23* to 26/25 J2 S239D/I332E seq 8/7 seq 22/21 8/7 fused 22/21 fusedto 24*/23* to 26/25 J3 I332E/G236A seq 8/7 seq 22/21 8/7 fused 22/21fused to 24*/23* to 26/25 J4 S239D/I332E/G236A seq 8/7 seq 22/21 8/7fused 22/21 fused to 24*/23* to 26/25 K0 — seq 10/9 seq 18/17 10/9 fused18/17 fused to 24/23 to 26/25 K1 I332E seq 10/9 seq 18/17 10/9 fused18/17 fused to 24*/23* to 26/25 K2 S239D/I332E seq 10/9 seq 18/17 10/9fused 18/17 fused to 24*/23* to 26/25 K3 I332E/G236A seq 10/9 seq 18/1710/9 fused 18/17 fused to 24*/23* to 26/25 K4 S239D/I332E/G236A seq 10/9seq 18/17 10/9 fused 18/17 fused to 24*/23* to 26/25 L0 — seq 10/9 seq20/19 10/9 fused 20/19 fused to 24/23 to 26/25 L1 I332E seq 10/9 seq20/19 10/9 fused 20/19 fused to 24*/23* to 26/25 L2 S239D/I332E seq 10/9seq 20/19 10/9 fused 20/19 fused to 24*/23* to 26/25 L3 I332E/G236A seq10/9 seq 20/19 10/9 fused 20/19 fused to 24*/23* to 26/25 L4S239D/I332E/G236A seq 10/9 seq 20/19 10/9 fused 20/19 fused to 24*/23*to 26/25 M0 — seq 10/9 seq 22/21 10/9 fused 22/21 fused to 24/23 to26/25 M1 I332E seq 10/9 seq 22/21 10/9 fused 22/21 fused to 24*/23* to26/25 M2 S239D/I332E seq 10/9 seq 22/21 10/9 fused 22/21 fused to24*/23* to 26/25 M3 I332E/G236A seq 10/9 seq 22/21 10/9 fused 22/21fused to 24*/23* to 26/25 M4 S239D/I332E/G236A seq 10/9 seq 22/21 10/9fused 22/21 fused to 24*/23* to 26/25 Full-length sequences of heavy andlight chains are listed in colums V and VI. (The sequences in column Vmarked with * refer to the IgG1 sequence of SEQ ID NOs 24 and 23(wild-type sequences) that have been modified to have thesubstitution(s) corresponding to column II, and the respectivemutation(s) in the coding DNA).

EXAMPLE 2 Chimeric mAb A0 Specifically Recognizes the CD37 Antigen

The specificity of MAb A0 for cellular CD37 is tested in a FACScompetition assay on Ramos Burkitt lymphoma cells (ATCC #CRL-1596).Cells are grown in tissue culture flasks (175 cm²) usingRPMI-1640+GlutaMAX supplemented with 10% heat-inactivated fetal bovineserum, 12.5 mM HEPES, 1 mM sodium pyruvat, 1% MEM non-essential aminoacids as culture medium. Cells are cultivated with an initial density of3×10⁵ cells/ml at 37° C. and 5% CO₂ in a humidified atmosphere for threedays. The cultures are maintained at a cell concentration between 3×10⁵and 1.8×10⁶/ml by sub-cultivation in a ratio of 1:6 with fresh culturemedium 2-3 times a week. For FACS competition analysis, theCD37-specific mAb HH1 (Santa Cruz) directly labeled with phycoerythrin(PE) is used at a concentration of 1 μg/ml. The antibody is preincubatedwith the unlabelled competitor antibody A0 for 10 min at 4° C. at theindicated molar ratio. Thereafter, 1×10⁵ Ramos cells are incubated for30 min with the antibody mixture on ice. Thereafter, cells are washedtwice in phosphate buffered saline (PBS), resuspended in FACS buffer andmeasured on a BD FACS Canto. Results of such an assay are shown inFIG. 1. Addition of a control human IgG1 antibody (Sigma IgG1 kappa) at20-fold molar excess does not significantly reduce the mean fluorescenceintensity (MFI) of Ramos cells. Addition of either unlabelled HH1antibody or A0 antibody at 20-fold molar excess almost completelyabrogated binding of the directly labeled HH1 antibody. This indicatesthat A0 and HH1 antibodies recognize identical or similar epitopes onRamos cells and compete for binding to cellular CD37 antigen.

EXAMPLE 3 Binding of Humanized Versions of mAb A0 to Cellular CD37Antigen

Humanized versions of A0 are tested for their binding to cellular CD37antigen by FACS analysis. Antibodies are added to Ramos cells at theindicated concentrations and allowed for binding for 30 min at 4° C.Thereafter, bound antibody is detected with PE-labelled goat-anti-humanIgG antibody (Sigma), cells are washed twice with PBS, and thereaftercells are resuspended in FACS buffer and analyzed by FACS on a BD FACSCanto. Examples are shown in FIGS. 2 and 3 (antibodies A, B, C, D, I orA, H, I, J, K, L and M, respectively; see Table 1). Several of thehumanized versions of A0 show similar binding to Ramos cells as theparental antibody A0, indicating that humanization does not reducebinding to cellular CD37 antigen.

EXAMPLE 4 FACS Scatchard Analysis of Humanized Versions of Chimeric mAbA0

The affinity of humanized versions of antibody A0 (designated B, C, D,H, I and K; see Table 1) to cellular CD37 antigen is determined by FACSscatchard analysis as described elsewhere (Brockhoff et al., 1994).Briefly, antibody dilutions are prepared in a 96 well plate startingwith 100-400 nM in the first well (80 μl), followed by 11 dilution steps(1:2, 40+40 μl). 50 μl of mAb dilutions are added to FACS tubes, 150 μlcells (0.8×10⁶/ml=1.2×10⁵ cells/tube) are added to each FACS tube. Cellsare gently mixed and incubated for 1 h on ice. Thereafter 50 μl FITCconjugated secondary antibody (conc. 15 μg/ml; mouse mAb anti-hu IgG allsubclasses, Zymed 05-4211) is added, mixed, and incubated for 30 min onice. 4 ml PBS ph7.2 containing 0.02% acid are added thereafter, cellsare pelleted and resuspended in 300 μl PBS pH 7.2 and subjected to FACSanalysis using a BD FACS Canto. All experimental steps are performed onwet ice, all antibody dilutions are made in PBS/0.5% BSA+0.02% acid.FACS calibration is performed using Quantum FITC MESF (Premix) HighLevel Beads (Bangs Laboratories). All samples are measured using thesame FACS parameters. The ratio of bound IgG versus free IgG iscalculated from MFI values at different antibody concentrations anddisplayed as scatchard blot. FIG. 4 shows the MFI/antibody concentrationrelationship of several humanized variants of A0. The results showsimilar binding to Ramos cells of some of the humanized versions as thestarting antibody, with dissociation constants (K_(d)) ranging from 2.15to 4.90 nanomoles/liter.

EXAMPLE 5 ADCC Activity of Humanized Versions of the Chimeric mAb A0

The ability of humanized versions of A0 (designated B, C, D, H, J, K;see Table 1) to mediate antibody-dependent cell-mediated cytotoxicity(ADCC) is assessed using Ramos cells as target cells and IL2-stimulatedhuman PBMCs as effector cells. Ramos cells (Burkitt's lymphoma; ATCC#CRL-1596) were purchased from ATCC. Cells are grown in tissue cultureflasks (175 cm²) using RPMI-1640+GlutaMAX supplemented with 10%heat-inactivated fetal bovine serum, 12.5 mM HEPES, 1 mM sodium pyruvat,1% MEM non-essential amino acids as culture medium. Cells are cultivatedwith an initial density of 3×10⁵ cells/ml at 37° C. and 5% CO₂ in ahumidified atmosphere for three days. The cultures are maintained at acell concentration between 3×10⁵ and 1.8×10⁶/ml by sub-cultivation in aratio of 1:6 with fresh culture medium 2-3 times a week. An aliquot ofthe cell culture at a cell density between 1.5×10⁶/ml and 1.8×10⁶/ml andgrowing in the log-phase is centrifuged (200×g, i.e. 1000 rpm) for 10min. Cells are washed once with washing medium (RPMI 1640 w/oL-glutamine) and pelleted (200×g, i.e. 1000 rpm; 10 min). Cell pellet isresuspended in assay medium [1% BSA in RPMI w/o L-glutamine] and cellcount is determined. Cell concentration is adjusted to 2×10⁵/ml.

Approximately 50-80 ml whole blood drawn from healthy donors is used forthe isolation of PBMC. 10 ml whole blood are diluted 1:3.6 with 26 mlHBSS (Hanks' Balanced Salt Solution w/o calcium and magnesium) in a 50ml tube. 18 ml diluted whole blood is layered on top of 12 ml Lymphoprep(Nycomed Pharma) in a 50 ml tube and centrifuged at 370×g (1400 rpm) for35 min. The mononuclear cells from the interface are aspirated andwashed first with HBSS (750×g, i.e. 1900 rpm; 10 min), then a secondtime with HBSS (300×g, i.e. 1200 rpm; 10 min) and at last with HBSS(160×g, i.e. 900 rpm; 10 min). The pelleted cells are gently resuspendedin culture medium/assay medium (10% heat-inactivated human AB serum inRPMI 1640 w/o L-glutamine) using a pipette and the cell count isdetermined in the cell counter. The PBMC concentration is adjusted to1×10⁷/ml. The freshly isolated PBMC (5×10⁵/ml) are maintained in culturemedium (RPMI 1640 w/o L-glutamine supplemented with 10% human AB serum)in a tissue culture flask (75 cm²) at 37° C. in CO₂ incubator overnight. On the following day cells are stimulated with hIL-2 at a finalconcentration of 1 U/ml for 3 further days. IL-2 stimulated PBMC areseparated from cell debris on a Lymphoprep gradient. The purified IL-2stimulated PBMC are suspended in culture medium/assay medium at aconcentration of 1×10⁷/ml.

The co-cultivation of effector cells with target cells in presence ofspecific or unspecific antibody is performed in duplicates ortriplicates in 96-well round-bottom microtiter plates in a final volumeof 200 μl assay medium per well consisting of 10% human AB serum and 1%BSA in RPMI in 1:1 ratio. First effector cells (freshly isolated PBMCcells in 100 μl 10% human AB serum in RPMI per well) are plated,followed by target cells and antibody solution diluted in 50 μl 1% BSAin RPMI. As a control, effector cells are cultivated in assay mediumalone (effector cell control) and target cells are cultivated either inassay medium alone (spontaneous lysis) or in assay medium supplementedwith 1% Triton X-100 (maximal lysis). The co-culture is incubated at 37°C. in a humid CO₂ incubator for 3 hours. At the end of the incubationcells are removed from the culture medium by centrifugation (200×g, i.e.1000 rpm; 10 min) at room temperature. Cell free supernatants (100μl/well) are transferred into corresponding wells of a 96-wellflat-bottom plate. To determine the LDH activity in these supernatants100 μl reaction mixture (freshly mixed 250 μl catalyst with 11.25 ml dyesolution) are added to each well and incubated 30 min at roomtemperature in the dark. Then the absorbance is measured as describedbelow.

Cytotoxicity Detection Kit (LDH; Roche) is used to measure ADCCactivity. The detection of cytotoxicity is based on the measurement ofLDH enzyme activity released from plasma membrane-damaged cells. LDHreleased into the culture supernatants reduces the tetrazolium salt fromthe kit to formazan. The absorption maximum of formazan dye is measuredat 490 nm against a reference wavelength of 650 nm in an ELISA platereader. To calculate percent cell mediated cytotoxicity five controlsare performed in each experimental setup.

-   Background control I (1): LDH activity contained in the assay    medium, which is subtracted from values (3) and (5).-   Background control II (2): LDH activity contained in 1% Triton-X100    in assay medium, which is subtracted from maximal LDH release values    (4).-   Spontaneous LDH release (3): LDH activity released from target cells    alone.-   Maximal LDH release (4): Maximum releasable LDH activity in the    target cells.-   Effector cell control (5): LDH activity released from effector cells    only.

To determine the percentage cell mediated cytotoxicity, the averageabsorbance of the triplicates or duplicates is calculated and thebackground is subtracted according to the manufacturer's instructions.In FIG. 5, the results from an ADCC assay using an E:T ratio of 25:1 andRamos target cells are shown. Antibodies are added in a concentration of30 ng/ml. Both the starting mAb and humanized versions thereof displaysimilar ADCC activity against the Ramos cells. In conclusion,humanization of anti-CD37 mAb A does not significantly alter its ADCCinducing capacity.

EXAMPLE 6 Pro-Apoptotic Activity of Humanized Versions of the ChimericmAb A0

The pro-apoptotic activity of mAb A0 (=A) and humanized versions thereof(B, C, D and I; see Table 1) is assessed by measurement of AnnexinV/PIpositive cells after incubation of Ramos cells with mAbs. Ramos cells(Burkitt's lymphoma; ATCC #CRL-1596) are received from ATCC. Cells aregrown in tissue culture flasks (175 cm²) using RPMI-1640+GlutaMAXsupplemented with 10% heat-inactivated fetal bovine serum, 12.5 mMHEPES, 1 mM sodium pyruvat, 1% MEM non-essential amino acids as culturemedium. Cells are cultivated with an initial density of 3×10⁵ cells/mlat 37° C. and 5% CO₂ in a humidified atmosphere for three days. Thecultures are maintained at a cell concentration between 3×10⁵ and1.8×10⁶/ml by sub-cultivation in a ratio of 1:6 with fresh culturemedium 2-3 times a week. An aliquot of the cell culture at a celldensity between 1.5×10⁶/ml and 1.8×10⁶/ml and growing in the log-phaseis centrifuged (200×g, i.e. 1000 rpm) for 10 min. Cells are washed oncewith washing medium (RPMI 1640 w/o L-glutamine) and pelleted (200×g,i.e. 1000 rpm; 10 min). Cell pellet is resuspended in culture medium andcell count is determined. Cell concentration is adjusted to 1×10⁶/ml.100 μl cell suspension per well are plated into 96-well round bottomplates. Antibodies are diluted in cell culture medium containing 10% FBSand 100 μl antibody solution are added per well. Cells are incubated for20 to 24 hrs at 37° C. in CO₂ incubator and thereafter stained withVybrant apoptosis assay kit #2. Alexa Fluor 488 labelled Annexin V andpropidium iodide solution are added to the cells and incubated for 15min in the dark. Thereafter cells are resuspended in 400 μl AnnexinVbinding buffer and subjected to FACS analysis using a BD FACS Canto. Thepercentage of AnnexinV positive/PI negative cells and AnnexinV/PIpositive cells is determined in two-dimensional dot blots using FL1/FL2channels. An isotype matched non-binding antibody (Sigma human IgG1) isused as negative control.

In FIG. 6, the pro-apoptotic effect of various humanized versions of mAbA on Ramos cells are shown. Cells are incubated with antibody at 10μg/ml for 24 hrs, the total percentage of AnnexinV positive cells (PIpositive and PI negative) is displayed. Parental mAb A shows potentpro-apoptotic activity. Surprisingly, humanized versions show asignificantly reduced number of AnnexinV positive cells compared to theparental mAb A, indicative for altered pro-apoptotic activity of thehumanized antibodies. In conclusion, humanization of MAb A leads to areduction of its pro-apoptotic activity in this experimental setting.

EXAMPLE 7 ADCC Activity of Fc-Engineered Versions of Chimeric mAb A0

ADCC activity of Fc-engineered versions of mAb A0 (designated A1, A2,A3, A4; see Table 2) is assessed using Ramos cells as target cells. ADCCassay is performed as described above (Example 5). The result of theexperiment is shown in FIG. 7. Fc-engineered versions of A0 show clearlyimproved potency and efficacy compared to the parent mAb A0. Certain Fcvariants show improvement in maximal lysis of up to 100% compared to theparent mAb and improvement in EC₅₀ of up to 10-fold compared to theparent mAb. In conclusion, introduction of specific Fc mutants stronglyincreases the ADCC activity of chimeric mAb A0.

EXAMPLE 8 ADCC Activity of Fc-Engineered Versions of mAb B0

ADCC activity of Fc-engineered versions of mAb B0 (designated B1, B2,B3, B4; see Table 2) is assessed using Ramos cells as target cells. ADCCassay is performed as described above (Example 5). Fc-engineeredversions of B0 show clearly improved potency and efficacy compared tothe parent mAb B0. Certain Fc variants show improvement in maximal lysisof up to 80% compared to the parent mAb and improvement in EC₅₀ of up to20-fold compared to the parent mAb. In conclusion, introduction ofspecific Fc mutants strongly increases the ADCC activity of humanizedmAb B0. The results of the experiments are shown in FIG. 8.

EXAMPLE 9 Pro-Apoptotic Activity of mAbs A0 and B0

The pro-apoptotic activity of mAbs A0 and B0 on Ramos cells before andafter cross-linking with anti-IgG mAb is displayed in FIG. 9. Theapoptotic assay is performed as described in Example 6, for antibodycross-linking an anti-human IgG antibody (γ-chain specific; Sigma) isadded to the antibodies in a ratio of 1:1 and incubated for 15 min at37° C. prior to adding to the target cells. In FIG. 9 the CD37-specificmAbs are added at a concentration of 1 μg/ml with and withoutcross-linking Chimeric mAb A0 is a potent inducer of apoptosis evenwithout cross-linking, this effect is significantly enhanced aftercross-linking of the mAb. Surprisingly, without cross-linking, thehumanized mAb B0 is completely devoid of pro-apoptotic activity, howevershows potent pro-apoptotic activity after cross-linking with anti-IgGAb. In conclusion, this experiment shows that pro-apoptotic activity ofa humanized version of mAb A0 can be restored after antibodycross-linking.

EXAMPLE 10 Pro-Apoptotic Activity of Fc-Engineered Versions of mAb A0

The pro-apoptotic activity of Fc-engineered versions of chimeric mAb A0on Ramos cells is assessed by AnnexinV/PI staining as described inExample 6. Parent1 antibody A0 and Fc-engineered variants A2 and A4 aretitrated over a concentration range from 0.1 to 10.000 ng/ml. As can beseen in FIG. 10, all 3 antibodies show similar pro-apoptotic activity.In conclusion, this experiment shows that Fc-engineering of mAb A0 doesnot alter its pro-apoptotic activity.

EXAMPLE 11 a) B Cell Depleting Activity of Fc-Engineered Antibodies A2and B2 in a Whole Blood Assay

The efficacy and potency of depletion of normal B cells from human bloodis assessed using a whole blood assay. In this assay format, the testantibody is added to EDTA-treated samples of human blood from healthyindividuals and subsequently, after 3 to 4 hrs incubation at 37° C., thenumber of B cells is quantitatively measured by a 4-color FACS assay. Bycomparison to buffer or IgG controls, the degree of B cell depletion bythe test agent can be calculated. Due to the presence of human IgGlevels and effector cells similar to the situation in human beings invivo, this assay type is considered of high relevance for predicting theeffect of the test antibodies in vivo.

A quantitative FACS assay is used to determine the number of B cellsand/or spiked Ramos cells in blood samples derived from healthyindividuals. Quantification is performed using BD Trucount tubes whichcontain a known number of fluorescent beads which serve as internalstandard for quantification of the cell population of interest. B cellsare identified by 4-colour analysis using 4 different CD markers(CD3/CD14/CD19/CD45) in combination with FSC/SSC analysis.

270 μl fresh blood per well is incubated in a 48 well plate togetherwith 30 μl antibody dilution (in PBS) or PBS (buffer control) induplicates. Samples are incubated for 4 h at 37° C. and thereafterimmediately placed on ice. 33 μl of CD marker master mix is added toTrucount tubes and 50 μl of the blood-antibody mixture is added. Samplesare vortexed and incubated for 15 min at room temperature. Thereafter450 μl of lysis buffer is added, vortexed, and incubated for additional15 min at room temperature. Samples are placed on ice and immediatelysubjected to FACS analysis using a BD FACS Canto™ Flow Cytometer.Evaluation of data is performed using the BD FACSDiva software (Version5.0.2).

Fc-engineered chimeric and humanized mAbs A2 and B2 show excellentpotency on normal B cell depletion with EC₅₀ values ranging from 0.15 to0.35 nM. The degree of normal B cell depletion ranges from 57% to 65%.Rituximab, a registered antibody used for the treatment of B-NHL, istested in parallel and yields significantly lower depletion of B-cellsin this assay format (FIG. 11 A).

b) Fc-Engineering Introduces Superior B Cell Depleting Activity of A0and B0 Compared to Rituximab

The effect of mAbs on B cell depletion in human blood derived fromhealthy individuals is assessed as described in a). The nonFc-engineered mAbs A0 and B0 show B cell depleting activity in the rangefrom 13% to 26%, similar to Rituximab. Fc-engineering results in adramatic increase of B cell depleting activity for both mAbs, with amean percentage of depletion of 75%. This clearly demonstrates thesuperiority of A2 and B2 compared to Rituximab (FIG. 11 B).

c) Antibodies A2 and B2 do not Deplete T Cells and Monocytes in WholeBlood Assays

The effect of A2 and B2 on T lymphocytes (CD3-positive) and monocytes(CD14-positive) is assessed in parallel to the effect on B lymphocytes.No significant change of either T cell numbers or monocyte numbers isobserved, whereas a significant reduction of the number of B cells isseen (FIG. 11C). This indicates that A2 and B2 specifically deplete Bcells from human blood.

EXAMPLE 12 Fc-Engineering Introduces Superior ADCC Activity Compared toRituximab

ADCC activity of the Fc-engineered version A2 of mAb A0 is assessedusing Ramos cells as target cells. ADCC assay is performed as describedabove (Example 5). The non Fc-engineered antibody A0 shows a maximallysis of Ramos target cells which is inferior to Rituximab, an antibodyspecific for CD20 which is an approved treatment for patients sufferingfrom B cell lymphomas. Surprisingly, Fc-engineering of A0 leads to aclearly improved potency and efficacy of A2 over Rituximab. Thisindicates, that at similar antigen densities of CD20 and CD37 on Ramoscells the Fc-engineered anti-CD37 mAb A2 shows clearly improved ADCCactivity than Rituximab (FIG. 12).

EXAMPLE 13 Lymphoma Cell Depleting Activity of Fc-Engineered AntibodiesA2 and B2 in a Whole Blood Assay

The efficacy and potency of depletion of Ramos cells, a Burkitt'slymphoma derived cell line from human blood is assessed using a wholeblood assay as described in Example 11. In a modification of the assay,Ramos tumor cells are spiked in about a tenfold excess compared toendogenous B cells into the whole blood matrix, and their depletion isalso monitored by FACS analysis. Fc-engineered chimeric and humanizedmAbs A2 and B2 show good potency on Ramos cell depletion with EC₅₀values ranging from 0.35 to 0.54 nM. The degree of Ramos cell depletionranges from 36% to 55%. Rituximab, a registered antibody used for thetreatment of B-NHL, is tested in parallel and yields significantly lowerdepletion of Ramos cells in this assay format (FIG. 13).

EXAMPLE 14 In Vivo Efficacy of Fc-Engineered Antibodies A2 and B2 inDisease-Related Model

The in vivo anti-tumor efficacy of mAbs A2 and B2 is assessed using aRamos Burkitt's lymphoma model in nude mice. CD37-positive Ramos cellsare injected subcutaneously into the flank of the animals and i.v.treatment of the animals started when tumors are established. A twiceweekly treatment schedule is chosen (q3/4d), two different doses (8mg/kg and 25 mg/kg) are tested in parallel. Both mAbs show significantanti-tumor efficacy with T/C values ranging from 0.2% to 26%. Nosignificant difference between the two dose levels and between the twoantibodies are observed. However, there is a trend towards betterefficacy in the high dose A2 treated animals, with T/C of 0.2% and 5/10complete tumor regressions. All treatments are well tolerated with noapparent weight loss. In conclusion, mAbs A2 and B2 showed significantanti-tumor efficacy in the Ramos Burkitt's lymphoma model, with maximumactivity already obtained at the 8 mg/kg dose level. The activity iscomparable to that of rituximab which is tested in parallel. It has tobe noted that the in vivo activity observed with the Fc-engineeredantibodies A2 and B2 may be underestimated since these mAbs areoptimized for interaction with human but not murine effector cells. Thisoptimized interaction, which leads to strongly improved ADCC activity invitro when using human effector cells (Example 8), is not reflected inthe mouse model used. However, the data obtained in this experiment(shown in FIG. 14) provide in vivo proof of concept of CD37 targetingand thus can be used for estimating the therapeutic dose in humans.

EXAMPLE 15 Correlation of Pharmacokinetic and Pharmacodynamic Effect ofA2 and B2 in Mice For Estimating the Therapeutic Dose in Humans

A correlation between the serum concentrations of A2 and B2 to theirpharmacodynamic effect is established in mice using the Ramos tumorxenograft model. These studies demonstrate that a dose of 8 mg/kg A2 andB2 (formulated in a citrate buffer: 25 mM Na-Citrate, 115 mM NaCl, 0.04%Tween 80, pH 6.0) causes significant retardation of tumor growth in thisaggressive s.c. (subcutaneous) tumor model using a standard q3 or 4dantibody dosing schedule in mice, thus indicating continued activitythroughout the dosing interval. Furthermore, pharmacokinetic data areestablished for the same dose.

Using this PK/PD association in mice, an estimated human dose can becalculated using published data for the clearance (CL) of humanizedantibodies in humans (Lobo et al., 2004).

Full calculation for A2:

-   -   Mean AUC(0-∞) after single dose of 8 mg/kg=6099 μg·h/mL.    -   Given AUC(0-∞) in mice=AUC(ss,τ) in mice, and        AUC(ss,τ)/τ=C(ave,ss).    -   C(ave,ss) in mice (for τ=84 hours)=73 μg/mL, which is presumably        equivalent to C(ave,ss) in men (for τ=168 h).    -   Since AUC(ss,τ) in men=D/CL, and using the humanized antibody        clearance (CL) range in humans reported by Lobo et al, 2004.:        CL=7 mL/h/70 kg to 15 mL/h/70 kg.    -   For 7 mL/h/70 kg: 168 hr×7=1176 mL×73 μg=86 mg.    -   For 15 mL/h/70 kg: 168 hr×15=2520 mL×73 μg=184 mg.

Therefore, for A2, the estimated weekly dose for a 70 kg human is in therange of 86 to 184 mg. Using the same assumptions as described above,the calculated estimated human weekly dose for B2 for a 70 kg human is189 to 404 mg.

EXAMPLE 16 Antibodies A2 and B2 Show ADCC Activity on Multiple MyelomaCells

The expression of CD37 on a panel of multiple myeloma cell lines isassessed by FACS analysis using antibodies specific for CD37. Cells areeither incubated with a directly fluorescently labeled anti-CD37antibody or an unlabeled CD37-specific antibody followed by a secondfluorescently labeled antibody directed against the primary antibody.The fluorescence activity of the labeled cells is measured with a FACSCanto Flow Cytometer (BD Biosciences) and the fluorescence intensity isrecorded as MFI using the FACS Diva Software. 6 out of 11 testedmultiple myeloma demonstrate cell surface expression of CD37 (FIG. 15).One cell line (RPMI 8226) is subsequently tested in an ADCC assay asdescribed in Example 5 using the CD37-specific antibodies A2 and B2.Both antibodies demonstrate potent ADCC activity on RPMI 8226 cells withEC₅₀ values in the range of 25 ng/ml and a maximal cell lysis of about20% (FIG. 16). This example demonstrates that CD37-positive multiplemyeloma cells are susceptible to ADCC mediated cell lysis using theCD37-specific mAbs A2 and B2.

FIG. 15 shows the FACS analysis of six multiple myeloma derived celllines for CD37 expression. The open curves indicate reactivity with theCD37-specific antibody, the filled curves represent the negative controlantibody.

EXAMPLE 17 Pro-Apoptotic Activity of Antibodies A2 and B2 onPatient-Derived CLL Cells

The pro-apoptotic activity of A2 and B2 is assessed on patient-derivedchronic lymphocytic leukemia (CLL) cells. Peripheral blood mononuclearcells (PBMCs) are prepared from a patient with diagnosed CLL, afterinformed consent according to the declaration of Helsinki has beenobtained. The primary CLL cells are purified from freshly collectedblood according to Ficoll-Paque® plus procedure (StemCell Technologies,Meylan, France) and stored at 4° C. in RPMI 1640 culture mediumcontaining 10% heat inactivated human AB serum (Sigma, France) untiluse. The culture medium for primary CLL cells is RPMI 1640 supplementedwith 2 mM L-glutamine and 10% of heat inactivated human AB serum. Forexperimental use, primary CLL cells are counted in a hemocytometer andtheir viability is assessed by 0.25% trypan blue exclusion. Theviability of CLL samples is more than 90%. Cells are incubated at 37° C.for 24 hours with the antibodies at 30 μg/ml and thereafter thepercentage of AnnexinV positive cells is determined as described inExample 6. As shown in FIG. 17, Fc-engineered antibodies A2 and B2 showstrong pro-apoptotic activity on the primary CLL cells with about 90%(A2) and 40% (B2) AnnexinV positive cells. Both mAbs are clearlysuperior to rituximab, a B cell-specific antibody approved for thetreatment of B-NHL. Mab A2 demonstrates also clearly superior activitycompared to alemtuzumab, an antibody approved for the treatment ofB-CLL.

EXAMPLE 18 Generation of a Transgenic Mouse Model in which theEndogenous CD37 Gene is Replaced by its Human Homologue

A targeting vector is constructed which contains the coding sequence ofhuman CD37 (BAC (bacterial artificial chromosome) IDs: RP11-433N13,RP11-50I11) flanked by non-translated sequences. This targeting vector(which contains, in addition, loxP sites flanking exons 3-4 and the neoselection marker flanked by frt sites) is then used for homologousrecombination, using mouse ES cells and standard technology to replaceexons 1-8 of the mouse genomic sequence with the human counterpartsequences, To this end, the C57BL/6N ES cell line is grown on amitotically inactivated feeder layer comprised of mouse embryonicfibroblasts (MEF) in DMEM High Glucose medium containing 20% FBS (PAN)and 1200 u/mL Leukemia Inhibitory Factor (Millipore ESG 1107). 1×10⁷cells and 30 g of linearized DNA vector are electroporated (Biorad GenePulser) at 240 V and 500 F. G418 selection (200 g/mL) started on d2.Counterselection with Gancyclovir (2 M) starts on d5 afterelectroporation. ES clones are isolated on d8 and analyzed by SouthernBlotting according to standard procedures, e.g. by the use ofradiolabelled DNA probes specific for the target gene after expansionand freezing of clones in liquid nitrogen. Transgenic animals are thengenerated by standard procedures known in the art, e.g. by blastocystinjection and subsequent generation of chimeric animals. Animalsheterozygous and homozygous for human CD37 are obtained by conventionalbreeding of chimeric and heterozygous animals, respectively. Thesuccessful knock-out of the murine CD37 gene and the knock-in of thehuman CD37 gene is monitored at the protein level using standardprocedures, e.g. FACS analysis of peripheral blood lymphocytes orimmunohistochemical analysis of tissue sections.

EXAMPLE 19 Generation of Surrogate Antibodies

Monoclonal antibodies specific for macaque CD37 are generated by geneticimmunization of mice and rabbits using the complete coding sequence ofthe macaque CD37 antigen (Acc. No. ENSMMUT00000020744). Specificantibodies are selected using recombinant HEK293 or CHO cells expressingthe macaque CD37 antigen, e.g. by standard ELISA or FACS techniques. Thevariable heavy and light chain coding sequences of these antibodies areretrieved by PCR cloning and used for generation of chimeric antibodies(as described in Example 1) which harbor the VH and VL region derivedfrom the murine or rabbit starting antibody and an Fc portion identicalto that of an antibody of the invention, e.g. A2 or B2. The binding andfunctional properties can be investigated by the use of assay systemswhich utilize macaque CD37 expressing cells as target cells, e.g. forbinding, FACS, Scatchard analysis, ADCC and apoptosis assays.Ultimately, the surrogate antibody is selected by virtue of its B celldepleting activity in Cynomolgus monkey blood in vitro.

EXAMPLE 20 Preparation of Clones for Producing the Antibodies

In order to prepare clones for producing antibodies of the invention,e.g. antibodies A2, A4, B2 or B4, the DNA molecule encoding the completeheavy chain, e.g. with the sequence shown in SEQ ID NO: 27, 31, 35 or39, respectively, is inserted into the eukaryotic expression vectordesignated pBI-26, encoding in addition the selection markerdihydrofolate reductase from hamster.

The DNA molecule encoding the complete light chain, depicted in SEQ IDNO: 29, 33, 37 and 41, respectively, is inserted into the eukaryoticexpression vector designated pBI-49, encoding in addition the selectionmarker neomycin phosphotransferase. The DNA sequences of the entireheavy and light chains are sequenced completely.

The hamster cell line CHO-DG44, grown in suspension in chemicallydefined media, is co-transfected with the eukaryotic expression vectorsfor the heavy and for the light chain of the antibodies, as describedabove. Transfected cells are selected in medium without hypoxanthine andthymidine and in the presence of the antibiotic G418. Subsequently,cells are subjected to stepwise selection and amplification usingincreasing concentrations of methotrexate (MTX). From the 800 nM MTXamplification step, a single cell clone is selected based on growthperformance and antibody production in spinner runs, and iscryopreserved in a Safety Cell Bank (SCB).

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1) An antibody molecule that binds to human CD37 and that is derivedfrom a) a murine monoclonal antibody that is defined by i) a variableheavy chain comprising the amino acid sequence shown in SEQ ID NO: 2;and ii) a variable light chain comprising the amino acid sequence shownin SEQ ID NO:4, or from b) a non-human antibody recognizing the sameepitope of human CD37 as the antibody defined in a) or recognizing anepitope that is close to or overlaps with said epitope; wherein saidantibody molecule is a chimeric or a humanized antibody. 2) An antibodymolecule of claim 1 that is a chimeric antibody defined by i) a variableheavy chain comprising the amino acid sequence shown in SEQ ID NO: 2;ii) a variable light chain comprising the amino acid sequence shown inSEQ ID NO:4, iii) constant heavy and light chains that are of humanorigin. 3) An antibody of claim 2, wherein i) the constant heavy chainis a IgG1 chain, and ii) the constant light chain is a kappa chain. 4)An antibody of claim 3, wherein said constant heavy chain i) comprisesthe amino acid sequence shown in SEQ ID NO:24 and wherein said constantlight chain ii) comprises the amino acid sequence shown in SEQ ID NO:26.5) An antibody of claim 1 that is a humanized antibody defined by a)CDRs contained within the variable heavy chain as shown in SEQ ID NO:2,b) CDRs contained within the variable light chain as shown in SEQ IDNO:4, c) frameworks supporting said CDRs that are derived from a humanantibody, d) constant heavy and light chains that are from a humanantibody. 6) An antibody of claim 5 comprising a variable heavy chainwith a sequence shown in SEQ ID NO:6. 7) An antibody of claim 6comprising a variable light chain with a sequence shown in SEQ ID NO:12,14, 16, 18, 20 or
 22. 8) An antibody of claim 5 comprising a variableheavy chain with a sequence shown in SEQ ID NO:8. 9) An antibody ofclaim 8 comprising a variable light chain with a sequence shown in SEQID NO:12, 14, 16, 18, 20 or
 22. 10) An antibody of claim 5 comprising avariable heavy chain with a sequence shown in SEQ ID NO:10. 11) Anantibody of claim 10 comprising a variable light chain with a sequenceshown in SEQ ID NO:12, 14, 16, 18, 20 or
 22. 12) An antibody of claim 1,wherein said antibody has one or more mutations in the Fc domain thatmodulate one or more effector functions. 13) An antibody of claim 12,wherein said modulation of effector function is an increase inantibody-dependent cell-mediated cytotoxicity. 14) An antibody of claim12, wherein said one or more mutations in the Fc domain is a singlesubstitution at position 332, numbered according to the Kabat EUnumbering index. 15) An antibody of claim 12, wherein said one or moremutations in the Fc domain is a combination of substitutions atpositions 239 and 332, numbered according to the Kabat EU numberingindex. 16) An antibody of claim 12, wherein said one or more mutationsin the Fc domain is a combination of substitutions at positions 236 and332, numbered according to the Kabat EU numbering index. 17) An antibodyof claim 12, wherein said one or more mutations in the Fc domain is acombination of substitutions at positions 236, 239 and 332, numberedaccording to the Kabat EU numbering index. 18) An antibody of claim 14,wherein said substitutions are I332E, S239D and G236A. 19) An antibodythat binds to human CD37 and has a heavy chain comprising the amino acidsequence of SEQ ID NO:28. 20) The antibody of claim 19, which has alight chain comprising the amino acid sequence of SEQ ID NO:30. 21) Anantibody that binds to human CD37 and has a heavy chain comprising theamino acid sequence of SEQ ID NO:36. 22) The antibody of claim 21, whichhas a light chain comprising the amino acid sequence of SEQ ID NO:38.23) An antibody that binds to human CD37 and has a heavy chaincomprising the amino acid sequence of SEQ ID NO:32. 24) The antibody ofclaim 23, which has a light chain comprising the amino acid sequence ofSEQ ID NO:34. 25) An antibody that binds to human CD37 and has a heavychain comprising the amino acid sequence of SEQ ID NO:40. 26) Theantibody of claim 25, which has a light chain comprising the amino acidsequence of SEQ ID NO:42. 27) A DNA molecule comprising a regionencoding the variable heavy chain of an antibody of claim
 1. 28) The DNAmolecule of claim 27, wherein said variable heavy chain encoding regionis fused to a region encoding a constant heavy chain of human origin.29) The DNA molecule of claim 28, wherein said human constant heavychain is IgG1. 30) The DNA molecule of claim 29, wherein said IgG1 isencoded by a sequence shown in SEQ ID NO:23. 31) The DNA molecule ofclaim 28, wherein said human constant heavy chain has one or moresubstitutions in the Fc region at position 332, 229 or 236, numberedaccording to the Kabat EU numbering index. 32) A DNA molecule comprisinga region encoding the variable light chain of an antibody of claim 1.33) The DNA molecule of claim 32, wherein said variable light chainencoding region is fused to a region encoding a constant light chain ofhuman origin. 34) The DNA molecule of claim 33, wherein said constantlight chain is a kappa chain. 35) The DNA molecule of claim 34, whereinsaid kappa light chain is encoded by a sequence shown in SEQ ID NO:25.36) An expression vector comprising a DNA molecule comprising a regionencoding the variable heavy chain of claim 1 and/or a DNA moleculecomprising a region encoding the variable light chain of an antibody ofclaim
 1. 37) A host cell carrying one or more vectors of claim
 36. 38) Ahost cell carrying an expression vector comprising a DNA moleculecomprising a region encoding the variable heavy chain of an antibody ofclaim 1, and a second expression vector comprising a DNA moleculecomprising a region encoding the variable light chain of an antibody ofclaim
 1. 39) The host cell of claim 37, which is a mammalian cell. 40) Amethod for producing an antibody, comprising transfecting a mammalianhost cell with one or more vectors of claim 36, culturing the host celland recovering and purifying the antibody molecule. 41) A pharmaceuticalcomposition comprising, as the active ingredient, one or more anti-CD37antibody molecules of claim 1, and a pharmaceutically acceptablecarrier. 42) The pharmaceutical composition of claim 41, furthercomprising one or more additional therapeutic agents. 43) Thepharmaceutical composition of claim 42, wherein said one or moreadditional therapeutic agents are selected from agents that target a Bcell antigen other than CD37. 44) The pharmaceutical composition ofclaim 43, wherein said B cell antigen is CD20. 45) The pharmaceuticalcomposition of claim 42, wherein said one or more additional therapeuticagents are selected from agents that induce apoptosis. 46) Thepharmaceutical composition of claim 45, wherein said agent is amodulator of a TRAIL receptor. 47) The pharmaceutical composition ofclaim 41 for depleting B cells that express CD37 on their surface. 48)The pharmaceutical composition of claim 47 for the treatment of B cellmalignancies. 49) The pharmaceutical composition of claim 48, whereinsaid B cell malignancy is selected from B cell non-Hodgkins lymphoma, Bcell chronic lymphocytic leukemia and multiple myeloma. 50) Thepharmaceutical composition of claim 47 for the treatment of autoimmuneor inflammatory diseases that involve B cells in their pathology. 51)The method of depleting CD37 expressing B cells from a population ofcells, comprising administering to said population of cells an antibodymolecule of claim 1 or a pharmaceutical composition containing suchantibody molecule. 52) The method of claim 51, which is carried out invitro. 53) A method for treating a patient suffering from a B cellmalignancy selected from B cell non-Hodgkins lymphoma, B cell chroniclymphocytic leukemia and multiple myeloma, comprising administering tosaid patient an effective amount of a pharmaceutical composition ofclaim 41.